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Published on 29 September 2016 on http://pubs.rsc.org | doi:10.1039/9781782628828-FP001
Bioinorganic Chemistry
Molybdenum and Tungsten Enzymes
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RSC Metallobiology Series
Editor-in-Chief:
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Titles in the Series:

1: Mechanisms and Metal Involvement in Neurodegenerative Diseases
2: Binding, Transport and Storage of Metal Ions in Biological Cells
3: 2-Oxoglutarate-Dependent Oxygenases
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5: Molybdenum and Tungsten Enzymes: Biochemistry
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Molybdenum and Tungsten

Enzymes
Bioinorganic Chemistry
Edited by
Russ Hille
University of California, Riverside, CA, USA
Email: russ.hille@ucr.edu
Carola Schulzke
University of Greifswald, Germany
Email: carola.schulzke@uni-greifswald.de
and
Martin L. Kirk
University of New Mexico, Albuquerque, NM, USA
Email: mkirk@unm.edu
Published on 29 September 2016 on http://pubs.rsc.org | doi:10.1039/9781782628828-FP001 View Online
RSC Metallobiology Series No. 6
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Preface
In the late 1950s and early 1960s, evidence was accumulating that molybde-
num was not simply present in the enzyme xanthine oxidase from cow's milk
but that it was required for its activity and changed its oxidation state in the
course of the reaction with substrate. In a tour-de-force isotopic substitution
study reported in Nature in 1966, R.C. Bray and L.S. Meriwether demon-
strated unequivocally that the EPR signals elicited by the enzyme upon treat-
ment with xanthine arose from a molybdenum-containing active site. It is
a happy coincidence but altogether fitting that this volume marks the 50th
anniversary of this seminal work.
For many years, only five enzymes were recognized as possessing molyb-
denum in their active sites: nitrogenase from bacteria such as Klebsiella
pneumoniae and Azotobacter vinelandii; xanthine oxidase from bovine milk
(and other vertebrate sources); aldehyde oxidase from vertebrate as well as
bacterial sources; the vertebrate sulfite oxidase; and the assimilatory nitrate
reductase from plants (and algae and fungi). That began to change in the
1980s with the demonstration by K. V. Rajagopalan that an organic cofactor
accompanied the molybdenum in the active sites of these enzymes (with the
exception of nitrogenase), and with the contemporaneous discovery that
tungsten was also found in the active sites of enzymes in certain bacteria.
There are now several dozen molybdenum- and tungsten-containing
enzymes that have been crystallographically characterized, along with most
of the enzymes responsible for the biosynthesis of the organic cofactor vari-
ously known as molybdopterin, tungstopterin and pyranopterin. The active
site metal centres of these enzymes have proven to be fascinating and chal-
lenging targets for synthetic inorganic chemists, and both enzymes and
synthetic models have proven fertile ground for the application of a range
of physicochemical and spectroscopic methods probing their physical and
electronic structures as well as their intrinsic reactivity. At present, well over

Molybdenum and Tungsten Enzymes: Bioinorganic Chemistry
Edited by Russ Hille, Carola Schulzke, and Martin L. Kirk
Published by the Royal Society of Chemistry, www.rsc.org
v
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vi Preface
50 molybdenum- and tungsten-containing enzymes have been isolated and
characterized, and these have been found to catalyze a broad range of oxi-
dation-reduction reactions, and even reactions that (at least formally) do
not involve oxidation–reduction of substrate. These enzymes are found in
a wide range of metabolic pathways and play particularly prominent roles in

the global cycling of nitrogen, sulfur and carbon. Many have vital roles in
bacterial bioenergetics, catalyzing crucial energy-conserving reactions
under a variety of growth conditions. Indeed, they seem to have been among
the earliest enzyme systems to have arisen, as reflected in their near-univer-
sal distribution in the biosphere. Finally, genomics analyses have led to the
identification of hundreds of genes encoding putative new proteins that are
likely to possess one or another metal. These systems represent an enormous
frontier of new enzymes that remains to be explored.
This title provides an up-to-date account of the state of our understanding
of molybdenum and tungsten enzymes and is divided into three volumes,
dealing with: (1) the enzymes themselves, along with pyranopterin cofac-
tor biosynthesis and incorporation of the mature cofactor into apoprotein
(Molybdenum and Tungsten Enzymes: Biochemistry), (2) inorganic complexes
that model the structures and/or reactivity of the active sites of each major
group of molybdenum and tungsten enzymes (Molybdenum and Tungsten
Enzymes: Inorganic Chemistry) and (3) spectroscopic and related methods of
physical chemistry (including computational work) that have been applied
to both enzymes and model compounds (Molybdenum and Tungsten Enzymes:
Physical Methods). Each volume is introduced by an overview chapter written
by a leading expert in the field, followed by the individual chapters that detail
specific topics associated with each volume. The intent of these overview
chapters is to provide an overarching and unifying theme that places each of
the three major subject areas in proper context.
We are deeply indebted to each of the contributors for their efforts, which
lay out the current state of our understanding in each of the many subject
areas considered. The coverage of these volumes is inevitably incomplete
due to space constraints, however, and for this we apologize. However, the
topics that are covered are presented to the reader in considerable detail;
written in a style and spirit that will be fully accessible by current researchers
in the field as well as those who wish to learn more about these fascinating
metalloproteins. We sincerely hope that these volumes will underscore how
rapid the progress has been over the past decade or so, and also how rapidly
the field is expanding. The ultimate goal is to stimulate further research on
molybdenum and tungsten enzymes, and especially to encourage new inves-
tigators to take up one or another aspect of these systems. It seems inevitable
that many exciting new discoveries lie in wait.
Russ Hille
Carola Schulzke
Martin L. Kirk
Dedication
It is all too fitting that these volumes dealing with the bioinorganic chemistry
of molybdenum and tungsten be dedicated to three outstanding chemists
whose contributions to the field over many years continues to inform, illumi-
nate and inspire: Richard H. Holm, C. David Garner and John H. Enemark.
Prof. Holm has over 500 research publications (cited over 35 000 times)
covering a wide range of nickel, iron and molybdenum chemistry (among
other transition metals). He is perhaps most widely recognized for studies,
beginning in the 1970s, that describe the synthesis and characterization
of iron-sulfur clusters. This work came to include modelling the M and P
clusters of nitrogenase, which perhaps provided the motivation to investi-
gate models of mononuclear molybdenum-containing enzymes. His molyb-
denum work achieved great success with the synthesis of MoO2 models for
enzymes of the sulfite oxidase, and later the DMSO reductase family, and the
characterization of their properties as oxygen atom transfer catalysts. A key
contribution was his use of bulky ligands to the metal that prevented µ-oxo

vii
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viii Dedication
dimerization, which had long stymied work in the field. He is Higgins Profes-
sor of Chemistry at Harvard University, a member of the National Academy
of Sciences and the recipient of many other awards.
Prof. Garner already had a strong track record in the synthesis of copper
and molybdenum complexes when, beginning in the late 1970s, he became

one of the first researchers to apply the then-new analytical method of X-ray
absorption spectroscopy not only to models of molybdenum enzymes but
also to the enzymes themselves. The discovery of thiolate-like sulfur, Mo=O
and Mo=S ligands to the metal in the active sites of enzymes such as sulfite
oxidase, xanthine oxidase and DMSO reductase was critical in establishing
the molybdenum coordination environment in these enzymes and greatly
focused efforts to synthesize accurate structural and functional mimics of
the enzymes. With over 300 publications (having over 8000 citations), he is
presently Professor Emeritus at the University of Nottingham and a Fellow of
the Royal Society. He is also past President of the Royal Society of Chemistry.
Prof. Enemark was already well recognized for his work on metal nitrosyls
and related systems when he began to exploit the tris-pyrazolylborate ligand
as a scaffold on which to construct and study MoO2 and MoO complexes.
This work led to the synthesis and characterization of the first model that
fully mimicked the catalytic cycle of oxotransferase enzymes such as sulfite
oxidase. Enemark also played an instrumental role in the work that led to
the first crystal structure of sulfite oxidase. Since that time, Enemark has
pioneered the application of pulsed EPR methods to molybdenum enzymes
and synthetic models of their active sites; work that has led to a deep under-
standing of not simply the physical but also the electronic structures of these
systems. With over 250 publications and 10 000 citations, he is Regents Pro-
fessor of Chemistry at the University of Arizona, a former Fulbright Scholar
and recipient of the Humboldt Research Prize, among other national and
international recognitions.
Contents
Chapter 1 An Overview of the Synthetic Strategies, Reaction

Mechanisms and Kinetics of Model Compounds Relevant
to Molybdenum- and Tungsten-Containing Enzymes 1
Carola Schulzke
Introduction and Overview 1
Chapter 2 Pterin-Inspired Model Compounds of

Molybdenum Enzymes 8
Sharon J. Nieter Burgmayer, Benjamin R. Williams,
and Partha Basu
2.1 I ntroduction 8
2.2 Unveiling the Pterin Component of Moco 10
2.2.1 The Pterin is Discovered 10
2.3 Development of Pterin-Inspired Models 15
2.3.1 First-Generation Models 15
2.3.2 Second-Generation Dithiolene Pterins
and their Complexes 33
2.3.3 Third-Generation Dithiolene Pterins
and their Complexes 43
2.3.4 Chemical Behavior of Dithiolenes Substituted
by Pterin and N-Heterocycles 53
2.4 Unresolved Questions and Current Objectives 57
Acknowledgement 59
References 59

ix
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x Contents
Chapter 3 Electron Transfer Mechanisms in Molybdenum
and Tungsten Model Compounds 68
Bholanath Pakhira, Rudra Sarkar, and Sabyasachi Sarkar
3.1 I ntroduction to Molybdenum and Tungsten 68

3.1.1 Role in Biology 69
3.2 Model Systems 70
3.3 Electron Transfer in Molybdoenzymes 78
3.3.1 Principle Involved in Electron Transfer
Reactions 80
3.4 Conclusion 87
References 88
Chapter 4 Comparative Kinetics of Enzymes and Models 94

Christian Fischer and Lina Fischer
4.1 A ctive Sites of Molybdenum and Tungsten

Enzymes 94
4.2 General Remarks on Michaelis–Menten Kinetics 96
4.2.1 Determination of Michaelis–Menten
Parameters 100
4.2.2 Michaelis–Menten Kinetics for Oxygen
Transfer Reactions – “Quo Vadis?” 103
4.3 Kinetic Aspects of Oxygen Transfer
Reactions – Enzymes vs. Models 106
4.3.1 Oxygen Transfer Reactions Mediated
by Enzymes 106
4.3.2 Studying Half-Reactions of Model
Complexes 107
4.3.3 Selected Historical Steps in Functional
Model Development 116
4.3.4 Half-Reaction Kinetics Applied to a
Two-Substrate Catalytic Cycle – A Simulation 121
4.4 Dedication and Acknowledgements 124
References 125
Chapter 5 Synthetic Models of the Nitrogenase Clusters 130

Sonny C. Lee
5.1 I ntroduction 130

5.2 The Nitrogenase Metalloclusters: A Synthetic
Perspective 131
5.2.1 The F-Cluster 132
5.2.2 The P-Cluster 132
5.2.3 The FeMo-Cofactor 133
5.3 Synthetic Considerations 134
5.4 F-Cluster Analogues: The All-Ferrous State 136
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Contents xi
5.5 M odeling the Nitrogenase Superclusters 137
5.5.1 Heterometallic Cores 137
5.5.2 Topological Analogues 141
5.5.3 Heteroligated Cores 148
5.6 Cluster Assembly Mechanisms:

Chalcogen-Labeled Cores 154
5.7 Status and Prospects 158
Abbreviations 158
Acknowledgements 159
References 159
Chapter 6 Synthesis of Mono- and Bisdithiolene Molybdenum and

Tungsten Model Compounds 166
Hideki Sugimoto

6.2 Model Compounds for the DMSOR Family 169
6.2.1 The MoVIO2 and MoIVO Couple 170
6.2.2 The MoVIO and MoIV Couple 176
6.2.3 MoVIS(Se-R) and MoIVS Couple 181
6.3 Model Compounds for the Sulfite Oxidase Family 182
6.4 Model Compounds for the Xanthine Oxidase Family 183
6.5 W-substituted Model Compounds for the
DMSOR and Xanthine Oxidase Families 185
6.6 Model Compounds for Tungsten-Dependent Enzymes 186
6.6.1 The WVIO(S) Center 186
6.6.2 The WVIS(Se-R) Center 188
6.6.3 The WVI(CO/CN)(SR) Center 189
6.6.4 The Unidentified W Center 189
6.7 Conclusion 190
References 190
Chapter 7 Models for the Xanthine Oxidase Family of Enzymes 194

Charles G. Young

7.2 Molybdenum Hydroxylases 195
7.2.1 Overview of the Enzymes 195
7.2.2 Key Discoveries Informing Model Studies 196
7.2.3 Active Sites and Model Targets 199
7.3 Active Site Components: Synthetic Background 200
7.3.1 General Challenges 200
7.3.2 The Chalcogenido Components: Background,
Synthetic Approaches and Reagents 201
7.3.3 The Mono(dithiolene) Component:
Background and Synthetic Approaches 207
7.3.4 Maintaining Mononuclearity Post-Synthesis 209
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xii Contents
7.4 M olybdenum Hydroxylase Models 211
7.4.1 Models of Enzymes Containing Oxosulfido
and Oxoselenido Active Sites 211
7.4.2 Models for the MoO(µ-S)Cu Active Site
of CODH 223
7.5 Conclusion 226
Dedication and Acknowledgements 227
References 228
Subject Index 239

Published on 29 September 2016 on http://pubs.rsc.org | doi:10.1039/9781782628828-00001
Chapter 1
An Overview of the Synthetic

Strategies, Reaction
Mechanisms and Kinetics of
Model Compounds Relevant to
Molybdenum- and Tungsten-
Containing Enzymes
Carola Schulzkea
a
Institut für Biochemie, Ernst-Moritz-Arndt Universität Greifswald,
Felix-Hausdorff-Straße 4, D-17487 Greifswald, Germany
*E-mail: carola.schulzke@uni-greifswald.de
Introduction and Overview

Bioinorganic model chemistry in general comprises synthesizing complexes
which resemble the natural active sites of metalloproteins with respect to
either structure or function or (preferably) both and investigating their struc-
tural and spectroscopic characteristics and/or reactivity, most often in com-
parison with results from biological samples. This is important for a detailed
understanding of the roles of metal, ligands and specific functional groups
in the processes taking place at the active sites.

1
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2 Chapter 1
The following chapters in Molybdenum and Tungsten Enzymes: Inorganic
Chemistry review advances in the field of molybdenum- and tungsten-de-
pendent oxidoreductases and nitrogenase by bioinorganic model chemistry,
or more precisely the synthetic and catalytic evaluation of model systems.
All this falls into the core expertise of two of the three outstanding scien-
tists to whom this book is dedicated. Both Richard H. Holm and C. David
Garner have contributed formidably to the field of molybdenum and tung-
sten model chemistry for the respective molybdenum cofactor (Moco) and
tungsten cofactor (Wco) bearing enzymes. Although with generally the same
goal, i.e. furthering the understanding of the electronic and steric specifics
of the respective active sites and their reactivity, the approaches of the two
groups were quite distinct. Holm and coworkers created model systems for
the immediate coordination spheres even of those enzymes that were most
difficult to mimic, their chemical composition being rather uncommon in
inorganic chemistry. Garner, often in cooperation with organic chemist John
A. Joule, designed ligand model systems mimicking more of the rather com-
plex natural ligand molybdopterin by synthesizing asymmetrically substi-
tuted dithiolene ligands bearing N-heterocyclic groups, even investigating
different tautomeric forms thereof; all this in relation to the pterin part now
presumed (based on protein structural findings) to play a substantial role
for the reactivity of the oxidoreductases. Current work by the community
attempts to unify these two approaches in order to accomplish the organic–
inorganic synthesis of increasingly accurate Moco and Wco models with a
perfect match still being elusive.
The authors of the following chapters have in their own work necessarily
been inspired by the synthetic foundations of this field laid by Holm and
Garner and some were even fortunate enough to have directly participated
as their coworkers.
Holm has in addition for many years been involved in nitrogenase model
chemistry. Pivotal work on the enzyme includes establishing the presence of
the Fe4S4-ferredoxins in the α2β2 complex by their extrusion and characteriza-
tion, utilizing 19F NMR spectroscopy for determining the magnetic moment
of molybdenum in the M-Cluster at ambient temperature and investigating
the binding of small molecules to the isolated M-Cluster. His synthetic con-
tributions comprise the earliest syntheses of Fe–Mo–S clusters, the exten-
sion of this initial work to clusters with high nuclearity, double cubanes
that served as topological models of the nitrogenase P-Cluster. This work
included investigations of the reactivity of these model systems, demonstrat-
ing for example that it is possible to transform the alternate nitrogenase sub-
strate acetylene to ethylene with model clusters. All this was indispensable
in developing a molecular description of the metal sites in the nitrogenase.
In his work, Holm always considered the “big picture”, meaning: instead of
getting lost in utmost detail, he continued to question and investigate with
perseverance the discrepancies observed between what was found in the pro-
teins and what could be achieved in the chemical lab, still a key question for
many areas of bioinorganic chemistry.
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An Overview of the Synthetic Strategies, Reaction Mechanisms 3

Today nitrogenase model chemistry has come quite a long way and some
amazing science has been carried out. Still, we remain quite far away from
being able to mimic the M-Cluster with all its features. This is partly due to
the fact that for decades chemists tried to model a molybdenum–iron cluster,
lacking an interstitial atom, now recognized to be carbon.

The structural model chemistry for both the nitrogenase and molyb-
dopterin-containing cofactors is now well developed. At the same time, there
are still important features of the active sites that await accurate modelling
by bioinorganic chemists. This is what keeps this field challenging and inter-
esting for the respective communities made up of scientists with vision and
perseverance.
As will be laid out in the following chapters with respect to molybdopterin
(MPT), a complex has been published by Sharon Burgmayer (co-author of
Chapter 2 in this volume), which is the closest structural MPT-focused model
for the active sites known today. There are only minimal differences (oxida-
tion state of the nitrogen atoms in the pyrazine part, for instance) to the nat-
ural molecule, although these may have an important impact on reactivity.
Incorporating this MPT model or another one developed by Partha Basu to
a molybdenum or tungsten centre which more closely resembles the actual
active site would be a fantastic achievement, although admittedly the respec-
tive chemistry is very difficult. In fact, a combination of model chemistry
focusing on mimicking molybdopterin and of the immediate coordination
sphere would actually involve merging Garner's and Holm's achievements
and constitutes the most challenging aim of the respective bioinorganic
community nowadays. Most likely it will be the active site of arsenite oxidase
that will be modelled in all detail first, since in its reduced form it resembles
molybdenum dithiolene complexes which are most common, i.e. mono-oxo
bis-dithiolene molybdates (iv). For any other enzyme, model chemistry is
more difficult as the active sites typically do not bear an oxo ligand together
with two molybdopterins in their reduced form. Therefore, in addition to
simply coordinating a more or less close molybdopterin model to molybde-
num (and tungsten) it will also be necessary to fine-tune procedures allow-
ing modification of such models, namely replacing the oxo ligand by ligands
mimicking coordination to the peptide (alcoholates, thiolates etc.), replacing
one dithiolene by a sulfido, oxo, hydroxide or hydrosulfido ligand, oxidiz-
ing the model to oxidation states v (intermediate in the catalytic cycle) and
vi (the opposite end of the catalytic process) and to make the compounds
water insensitive and water soluble. All this has been done before with com-
plexes with simpler dithiolene ligands or with other ligand systems. Once
the functional groups of MPT are present, however, the respective chemis-
try becomes unknown ground and very difficult. And this is not all. Even
though a dithiolene ligand with all the functional groups of MPT is requi-
site for allowing detailed structure–function relationships to be studied, it
will probably not be enough. In order to really understand the distinct func-
tion of a specific structural motif of MPT it will be necessary to compare the
electronic states, the catalytic potential and the stability of models with and
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4 Chapter 1
without this specific motif. Eventually, the bioinorganic chemists' efforts will
have to be united with those of the biologists. The models have to be tested
with regard to being incorporated into the apoenzymes or recognized by the
respective chaperones that can insert them into the enzymes.
Semisynthetic enzymes will have to be prepared and compared for their

activity and need to be spectroscopically characterized. The requirements for
models in a biological environment are high with respect to stability and
solubility; another challenge for the bioinorganic chemist.
None of the above is trivial work and it is certainly impossible for one group
only to address all these issues. Even though the community is not huge, the
determination of its members is and the work of many contributors in this
field is heading in one or another of the directions described above.
Only when looking at all the results of all the individual contributors to
this field and combining these insights can we understand and describe the
inorganic chemistry of the respective active sites really accurately.
As many cooperative efforts are required, the specific fields laid out in the
following chapters, intended to inform in all needed detail, cannot be viewed
completely independently from each other. Consequently there will be some
overlap between the individual chapters, which should still provide a suit-
able reading experience for all types of readers.
In Chapter 6, Hideki Sugimoto reviews model systems for molybdenum
and tungsten oxidoreductases which bear one (mimicking sulfite oxidase
family, xanthine oxidoreductase family) or two (mimicking DMSO reductase
family and tungsten enzymes) dithiolenes, and focuses to some extent on
the first and second coordination shell of the enzymes. Sugimoto published
the first structurally characterized model with dithiolene ligands bearing
the pyrane ring of MPT, extending the respective model chemistry a bit far-
ther out and contributing some interesting and detailed studies about the
electronic properties of a variety of dithiolene model systems. Dithiolene
ligands are special as they are non-innocent ligands, and can directly partic-
ipate in oxidation–reduction reactions. This also means that it is not trivial
working with these fascinating molecules. Both Garner and Holm developed
procedures for the synthetic chemistry which are still widely applied today
and without which there would hardly be any structural model chemistry of
molybdenum and tungsten enzymes possible.
In Chapter 2, Partha Basu and Sharon Burgmayer detail the progress model
chemistry has made with a focus on all the features of molybdopterin. Basu
was a postdoc with John Enemark, the third outstanding scientist to whom
this book is dedicated, whose work has focused on spectroscopy as much as
synthesis. Burgmayer has received training in the group of Ed Stiefel, another
founding father of the respective bioinorganic chemistry, and she was intro-
duced to pterins early on in her career. Both Basu and Burgmayer have for
many years, both independently and in collaboration, furthered the model
chemistry of N-heterocyclic dithiolene chemistry. Burgmayer has devel-
oped the molybdenum bound model system which is most closely related
to the natural molybdopterin today and is deeply involved in bringing pterin
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chemistry together with pyrane and dithiolene chemistry. Basu follows dif-
ferent, complementing approaches towards the same goal and along the way
managed to exploit the interesting chemistry these electronically flexible
systems provide, for instance in the field of heavy metal ion sensing. Pterin
chemistry per se is well understood, although elaborating pterins with pyrane

and dithiolene moieties becomes incredibly complicated.
In Chapter 7, Charles G. Young outlines the bioinorganic efforts to under-
stand enzymes of the xanthine oxidase family. These enzymes distinguish
themselves from the other families in that (1) they do not catalyze the typical
oxygen atom transfer reaction but rather the insertion of an oxygen atom
into a C–H bond, i.e. they are hydroxylases; and (2), they bear a sulfido ligand
in the active site which is crucial for catalytic activity. Young has worked
for decades in this field and designed many model systems which have fur-
thered our understanding of the sulfido ligand's role for the electronic states
of the central metal and the complexes' (and hence enzymes') reactivity. The
respective chemistry is very difficult, particularly when trying to address all
ligands of the natural cofactors. Most often, for example, an auxiliary ligand
system in place of the dithiolenes is used for stabilizing the respective com-
plexes allowing the detailed investigations on the sulfide ligand's relevance
to be carried out.
Young has extensively cooperated with many in the area of bioinorganic
spectroscopy, including John Enemark, with whom he was a postdoc.
With respect to the catalytic evaluation of model compounds of the molyb-
denum and tungsten cofactors, it was Holm who first characterized a model
system catalyzing oxygen atom transfer, i.e. the oxidation of triphenylphos-
phine by dimethyl sulfoxide. This model reaction is now an indispensable
tool for the bioinorganic Moco/Wco community. The reaction takes place
at ambient conditions only when catalyzed and it can be nicely followed by
phosphorous NMR. Although DMSO actually is a natural substrate (as the
DMSO reductase family evidences), triphenylphosphine is not and recent
and current efforts have focused on more physiologically relevant reac-
tions with the most difficult task of employing water as solvent and/or sub-
strate. The authors of the two chapters dealing with catalysis and kinetics
are involved in the respective developments. In particular, Sabyasachi Sarkar
has been responsible for some of the most exciting findings in this field by
employing a comparatively simple model system which turned out to be a
fabulous work horse.
In Chapter 3, Sabyasachi Sarkar describes the achievements that were
made in applying model systems in catalytic processes mimicking natural
enzymatic substrate transformations and in understanding the respective
mechanisms in detail. Among other achievements in this area he discovered
the catalytic potential of comparatively simple dithiolene-bearing model
compounds employing maleonitrile ligands [MOx(mnt)2]2− (M = Mo, W; x
= 1, 2). He coaxed some surprising substrate transformations out of these
small complexes by fine-tuning the respective reaction conditions. Reactions
described by Sarkar and coworkers include transformation of hydrogensulfite
View Online
6 Chapter 1
to hydrogensulfate relevant for sulfite oxidase, the reduction of hydrogencar-
bonate to formate at pH 7.5, i.e. basically transformation of CO2 and relevant
for tungsten-formate dehydrogenase, the oxidation of aldehydes to carbox-
ylic acids relevant for aldehyde oxidase and the transformation of acetylene
to acetaldehyde relevant for the extraordinary tungsten-dependent acetylene

hydratase plus various non-natural substrates like phosphanes.
Sarkar too has cooperated with John Enemark in the past, emphasizing
that many specialists with different expertise are needed in order to under-
stand the molybdenum and tungsten oxidoreductases in detail, in particular
when it comes to reactivity.
In Chapter 4, Christian Fischer reviews catalytic properties of models and
enzymes more specifically. This chapter includes a detailed kinetic evalua-
tion of the catalytic systems. As a young academic Fischer has only recently
started working in the field of molybdenum- and tungsten-dependent
enzymes. He has graduated from the Leibnitz Institute of Catalysis led by
Matthias Beller and is an expert in the field of catalysis. His involvement will
hopefully extend what we know about the kinetics of models and enzymes
continuing Holm's excellent work in this field. Notably, Fischer has already
developed and investigated the first catalytic system being able to operate in
pure water.
In the case of the bioinorganic chemistry relevant for nitrogenase, the
determination of the presence of an interstitial carbon at the centre of the
M-Cluster of nitrogenase has fuelled much new model chemistry. At present,
there are only three variants known of the M-Cluster with vanadium or iron
in place of the molybdenum but no variation of the general cluster structure
or coordination environment. Therefore structural models are rather scarce,
particularly when compared with the Moco or Wco chemistry. When it comes
to functional models, the achievements in particular of the last years are
impressive. Many molybdenum systems and now even a few iron complexes
are known which can catalytically reduce the rather inert elemental nitrogen
to ammonia. These model systems are intended to mimic the natural process
because it would be industrially immensely important being able to split the
nitrogen triple bond catalytically at mild conditions. More importantly, from
a bioinorganic point of view it would be very important to reliably identify the
place on the M-Cluster where nitrogen is bound and transformed. Although
recent work of Dean, Hoffman and Seefeldt strongly supports binding of N2
on the Fe2,3,6,7 face of the M-Cluster, nitrogenase has still not been “caught
in the act” of nitrogen binding crystallographically. Advocates of molybde-
num- and iron-based chemistry have been fighting over this for decades and
we still do not know with absolute certainty what happens at the active site of
this intriguing, long-known and yet mysterious enzyme. In Chapter 5, Sonny
Lee, a former coworker of Holm in this particular field, has taken on the huge
task to guide the reader through the various fundamental and recent devel-
opments in the bioinorganic model chemistry of nitrogenase.
The work of each of the authors contributing to this volume has been
seminal with respect to developing the most accurate models of molyb-
dopterin and for the structures and functions of the molybdenum- and
View Online

tungsten-containing centres of nitrogenase and oxidoreductase, the best
of the latter being able to catalyze biologically relevant reactions. Most of
the authors have been directly involved in the outstanding achievements of
those this book is dedicated to.
I am thankful to all of them for having expertly detailed the fundamental

and the recent advancements in the field of molybdenum and tungsten bioi-
norganic chemistry. Further, I am immensely grateful to C. David Garner and
Richard H. Holm in particular, and also to John Enemark for having inspired
me at the beginning of my independent academic work to an extent that it
was actually their work that made me choose my scientific home in this field
of research.
Chapter 2
Pterin-Inspired Model
Compounds of Molybdenum
Enzymes
Sharon J. Nieter Burgmayer*a, Benjamin R. Williamsa,
and Partha Basu*b†
a
Department, of Chemistry, Bryn Mawr College, Bryn Mawr, PA 19010, USA;
b
Department of Chemistry and Biochemistry, Duquesne University,
Pittsburgh, PA 15282, USA
*E-mail: sburgmay@brynmawr.edu, brwilliams@brynmawr.edu
2.1 Introduction
The molybdenum cofactor (Moco) is an extraordinary molecule in biology. As
a small metal-containing compound, it has the unprecedented combination
of a dithiolene chelate for metal binding and a pterin appended to a pyran
ring. The resultant cofactor is electronically nimble due to the presence of
three redox active moieties, i.e. the molybdenum atom, the dithiolene and
the pterin, which in concert can support a range of redox events.
The long history of modeling Moco dates back nearly 50 years.1 The early
models were developed before the pterin in Moco was identified and before
†
Present address: Department of Chemistry and Chemical Biology, Indiana University Purdue
University at Indianapolis, Indianapolis, IN 46202, Email: basup@iupui.edu.

8
Pterin-Inspired Model Compounds of Molybdenum Enzymes 9
a structure of Moco had been determined by X-ray crystallography. Yet these
early models provided a base of knowledge from which an understanding of
possible mechanisms and of the electronic environment within the Mo inner
coordination sphere was built. The chapters in this second volume discuss
the contributions made by others that enhanced our knowledge of the func-
tion of molybdenum and tungsten enzymes.
The discovery of the pterin-substituted dithiolene ligand in Moco estab-
lished a specific target for modeling chemists, albeit a target of considerable
synthetic difficulty. There have been concerted efforts to reproduce aspects
of the pterin-dithiolene ligand in Moco and their synthetic model systems
have evolved over the past 30 years.1a,2 The complexity of pterin and pyra-
nopterin chemistry starts with the different numbering systems used, and
these are shown in Figure 2.1 for the pterin and pteridine ring systems, the
fully reduced, open form initially proposed for Moco and two numbering sys-
tems used for the reduced pyranopterin forms of Moco.3
Figure 2.1
Numbering systems for pterin, a derivative of the pteridine ring sys-
tem (top), in an uncyclized and fully reduced form initially proposed
for Moco (middle) and as the cyclized and reduced pyranopterin form
observed by crystallography in the majority of molybdenum enzymes
(bottom).
10 Chapter 2
The scope of this chapter is to review the model work directed towards
the pterin aspects of the molybdenum cofactor. Our objective is to provide
a detailed description of model work involving pterin, pteridine and related
N-heterocycle systems that have been specifically developed to mimic fea-
tures of Moco, as well as the study of these molecules in the absence of metal.
Because the evolution of pterin models parallels the progress of revealing the
pterin component of Moco, the chapter begins with a review of the key devel-
opments that unveiled the pterin ligand of Moco.
2.2 Unveiling the Pterin Component of Moco

In this section, an overview is of studies presented that revealed the pres-
ence of a pterin in molybdenum enzymes and eventually the structure of the
pterin–dithiolene ligand of Moco. This background provides the context for
pterin-inspired model work over the past three decades and highlights how
the making of synthetic models for metal sites in enzymes follows acquisi-
tion of experimental data obtained from the metal site in the proteins.
Prior to the discovery of the molybdopterin, it had already been established
that the molybdenum catalytic center of the molybdenum enzymes was part
of a dissociable cofactor. Reconstitution of a Moco-deficient mutant of Neu-
rospora crassa nit-1 by the dissociated molybdenum cofactor from a variety
of molybdoenzymes regenerated nitrate reductase activity in nit-1 further
confirming that molybdenum and a particular ligand set comprised Moco.4
Spectroscopic studies, primarily electron paramagnetic resonance (EPR) spec-
troscopy and extended X-ray absorption fine structure (EXAFS) spectroscopy,
had suggested both oxo and sulfur coordination in the dissociated Moco sam-
ples,2b,5 but the pterin component remained invisible in these studies. It was
not until 1980, when fluorescence was detected in degraded Moco-containing
protein samples, that an indication of the presence of a pterin emerged.6
2.2.1 The Pterin is Discovered

The investigation of the pterin component of Moco was a focus of Rajago-
palan and his coworkers.7 Initially, various oxidative treatments of Moco
were shown to yield different pterin species, each having distinctive fluores-
cence and electronic spectral signatures, and each pterin species substituted
at the C6 position on the pterin system. One of these degradation products,
Form B (1), was proposed to have a thiophene structure similar to urothi-
one (2). Urothione was found to be a metabolite of Moco in humans.8 The
sulfur atoms in 2 became the starting point for hypothesizing a dithiolene
chelate attached at C6, and the dithiolene hypothesis was eventually con-
firmed through alkylation and trapping the dithiolene-substituted pterin.9
The trapped pterin-dithiolene molecule also provided further support for the
reduced state of the pterin. A tetrahydropterin had been suspected from the
lack of fluorescent behavior of Moco, whereas oxidative degradation of Moco
containing enzyme produced highly fluorescent oxidized pterins. A pictorial
summary of the pterin studies is shown in Figure 2.2.
Figure 2.2 summary of key experiments that revealed the pterin and dithiolene
A
groups, and led Rajagopalan to propose the bottom structure for Moco.
12 Chapter 2
In 1982, the culmination of these studies led to the proposal that the hid-
den ligand on Moco was a reduced tetrahydropterin substituted at C6 by
a four-carbon chain having a dithiolene chelate at the α- and β-carbons, a
hydroxyl group at the γ-carbon and terminated with a phosphate (Figure 2.1,
center).8,10 This ligand was initially named molybdopterin for “the special
ligand on molybdenum”, abbreviated as MPT. Since then, other names have
been sought and used for this ligand, especially since the pterin-dithiolene
ligand also is the special ligand in tungsten enzymes.3,11
The tetrahydropterin–dithiolene ligand generated considerable excite-
ment and speculation. It was unique in biochemistry, and is unusual in
chemistry. While dithiolenes were well-known ligands for molybdenum
and other metals, this was the first time a dithiolene was proposed to play
a role in biochemistry. On the other hand, tetrahydropterins were already
known molecules in biochemical processes, such as the tetrahydrobiop-
terin cofactor used by aromatic amino acid hydroxylases and tetrahydro-
folate in C1 transfer in methionine synthesis (Figure 2.3). Certainly, this
was the first time a pterin was found to be in combination with a dithio-
lene anywhere in chemistry.
The known redox roles of tetrahydropterins in biochemistry led Rajagopalan
to investigate the redox behavior of the pterin unit of Moco. They titrated Moco
within molybdoproteins (XO and SO) with two different oxidants, ferrocyanide
and the redox dye dichlorophenol indophenol (DCIP),12 and obtained unex-
pected results: two electron equivalents of either oxidant produced the spec-
tral signature of a fully oxidized pterin (Scheme 2.1), a result only consistent
with the pterin in Moco starting at the dihydro oxidation state rather than the
tetrahydro state as initially proposed. The interpretation at the time was that
the pterin in Moco, instead of the initially proposed tetrahydropterin struc-
ture, was a dihydropterin in an unusual tautomeric form.
Prior to the discovery of the pterin portion of Moco, it was assumed that
all molybdenum enzymes (except nitrogenase) used the same cofactor, since
Figure 2.3
Structures of other pterins used in biochemical catalysis.
Scheme 2.1 The results of redox titration experiments on molybdoenzymes.
Figure 2.4
Proposed structure of MGT with appended guanosine nucleotide.
isolated Moco solutions could regenerate nitrate reductase activity in the Neu-
rospora crassa nit-1 mutant.4b,4c Krüger et al.13 challenged the notion that the
proposed Moco in Figure 2.2 was the universal structure in all molybdoen-
zymes in 1986 when they proposed a larger structure with a second phos-
phorylated aromatic unit. Rajagopalan's group's definitive analytical work
on DMSOR from Rhodobacter sphaeroides proved that it possessed a modified
MPT, described as a phosphoric anhydride of MPT and 5ʹ-GMP (Figure 2.4),
which they called MGD for molybdopterin guanosine dinucleotide.14 They
noted that the relationship of MPT to MGD was analogous to that between
FMN and FAD.7 By 1992 additional variants of MPT with adenine cytosine,
inosine dinucleotides were identified. Dinucleotide substitution on the side
chain is restricted to Moco from prokaryotic sources.
After years of speculation, in 1995, the unique structure of molybdopterin was
revealed by X-ray crystallography, first in the tungsten enzyme, aldehyde ferre-
doxin oxidoreductase isolated from the hyperthermophile Pyrococcus furiosus,15
and later as part of Moco in aldehyde oxidoreductase isolated from Desulfovib-
rio gigas.16 These structures confirmed most of Rajagopalan's proposal with
one exception: both structures showed that the dithiolene formed a third ring,
a six-membered pyran ring fused to the pterin structure, presumably through a
cyclization reaction involving the side chain hydroxyl group (Figure 2.5). Since
these first two structures of Mo and W proteins were reported, dozens of X-ray
structures have been determined and all the structures have shown the same
pyranopterin–dithiolene structure for molybdopterin, with two exceptions. The
first exception was observed in 2003 for a membrane bound dissimilatory nitrate
14 Chapter 2
Figure 2.5
The pyranopterin structure within MPT determined by protein X-ray
crystallography of aldehyde oxidoreductase from Desulfovibrio gigas
(PDB 1DGJ, guanosine not shown).
Figure 2.6
Structure of Moco found in E. coli dissimilatory nitrate reductase.
reductase (Nar) isolated from Escherichia coli.17 Moco in this protein has two MPT
ligands displaying two different structures (Figure 2.6). The proximal MPT, i.e.
the pterindithiolene closest to the Fe4S4 cluster involved in the electron transport
chain, has the expected pyranopterin structure, but the distal MPT no longer has
a pyran ring. Pyran ring cleavage had been shown to be a facile reaction in a model
compound (see section 2.3.3.4) and this reaction was considered a possible role
for the MPT ligand. In this regard, the Nar structure was pivotal in providing evi-
dence that ring opening might occur in the protein. Soon after, a second example
of a cleaved pyran structure at the distal MPT was available in the X-ray structure
of ethylbenzene dehydrogenase isolated from Aromatoleum aromaticum.18
In 1993, Wuebbens and Rajagopalan reported a molecule called precur-
sor Z (3)19 as a biosynthetic precursor of Moco.20 This was the first stable
intermediate identified in Moco biosynthesis, and soon afterwards the
involvement of GTP in Moco biosynthesis in E. coli was reported.21 In 1997
a parallel scheme for plants was reported by Mendel et al.22 While several
structures for 3 were proposed in the literature, in 2004, based on 1H NMR data
Figure 2.7
Structures of the biosynthetic precursor to MPT, precursor Z, and com-
pound Z as the first isolated form of precursor Z.
Santamaria-Araujo et al. proved that 3 was a pyranopterin in the reduced state

with a geminal diol formed by hydration at the C1ʹ position (Figure 2.7).23 By
2006 a more complete picture of Moco biosynthesis emerged.24
2.3 Development of Pterin-Inspired Models

Pterin-inspired models have evolved in response to the acquisition and inter-
pretation of new data from Moco, both from holoenzymes and from dis-
sociated, isolated Moco samples. The key events punctuating the growing
knowledge of the nature of the pterin in Moco were described in the preced-
ing section and are graphically presented as a timeline in Figure 2.8. The
development of pterin models can be appreciated and understood when
compared to this timeline. For the purposes of this chapter, the pterin model
evolution presented below is partitioned into three sections, described as
three generations of models.
2.3.1 First-Generation Models

The earliest period of work on pterin models for Moco followed the discovery
of the pterin unit within Moco, and occurred prior to the confirmation of
the dithiolene chelate. These early studies explored the coordination chem-
istry between molybdenum and pterins or other structurally related mole-
cules such as pteridines (Figure 2.1, top). The resulting themes of this body
of work include the favorable coordination by molybdenum in several oxida-
tion states to the O4, N5 chelate site in pterin (see Figure 2.1 for numbering),
a variety of reactivities exhibited by MoVI-tetrahydropterin systems and the
highly delocalized electronic structures in molybdenum–pterin complexes
that defy formal oxidation state assignments to Mo and pterin.
2.3.1.1 Models Where Molybdenum is Coordinated Directly to

Pterins
The pterin component of Moco has been fascinating – why did Nature make
such a complicated heterocyclic substituent for the dithiolene tether to molyb-
denum? The original molybdopterin structure (Figure 2.1, center structure)
16 Chapter 2
Figure 2.8
Timeline illustrating the progress in defining the identity of the
pterin ligand in Moco, and the development of selected pterin model
compounds.
proposed by Rajagopalan, suggested several metal binding sites in addition

to the pterin–dithiolene (Figure 2.9) and prompted the notion that the pterin
could present alternative coordination environments for molybdenum.
The first example of pterin coordination to Mo was reported in 1986.
Xanthopterin under basic conditions reacted with molybdate to produce a
Figure 2.9
The proposed structure of Moco after Rajagopalan, and alternative
molybdenum binding modes to molybdopterin.
Figure 2.10
Ball-and-stick drawing of a pterin chelating molybdenum complex
[Mo2O5(xanthopterinate)2]2−. Red, oxygen; green, molybdenum; blue,
nitrogen; grey, carbon.
dimeric species [Mo2O5(xanthopterinate)2]2− (Figure 2.10) where, amusingly,

the yellow butterfly pigment xanthopterin produced a dimeric Mo2-bis(xan-
thopterinate) compound with a butterfly conformation of the two bridging
pterin chelates.25 The significance of this system was that MoVI in the biolog-
ically relevant form of molybdenum [MoO4]2−, reacted with a pterin chelated
through the O4, N5 donor atoms. In the specific case of xanthopterin, a sec-
ond carbonyl functionality serves as a bridge to another Mo atom to struc-
turally reinforce the common [Mo2O5]2+ core. Although free xanthopterin
fluoresces strongly as is typical of oxidized pterins, coordination to Mo com-
pletely quenches its inherent fluorescence.
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“Oh, don’t shout like a cheap skate,” answered Ned disgustedly.
“Go and fix yourself up, if you can, so I won’t be ashamed to go to
supper with you!”
Laurie glared, swallowed hard, and finally nodded. “Listen,” he
said slowly. “You don’t have to be seen with me if it offends your
delicate sensibilities. Get it? And, what’s more, I don’t want to be
seen with you. I’m particular, too, you big bluff. When you want to go
to supper, you go!”
Laurie grabbed wash-cloth and towel, strode across the room, and
slammed the door resoundingly behind him. Left alone, Ned
shrugged angrily. “Ugly-tempered brute,” he muttered.
When supper-time came he descended alone to the dining-hall.
Laurie had not returned to the room. Laurie arrived a few minutes
late, with Kewpie, and took the seat at Ned’s left in silence. He had
put talc powder over the abrasion on his cheek-bone, and at a little
distance it would not have been noticed. Nearer, however, the lump
was plainly visible and seemed to be still swelling. Ned caught a
glimpse of it from the corner of his eye, but his irritation still
continued, and he offered no comment.
After supper both boys returned to No. 16, although not together,
and for two hours occupied opposite sides of the table, and
crammed for their last examination, which was due at ten to-morrow.
Neither spoke once during the evening. At nine Laurie closed his
books and went out. Half an hour later Ned undressed and went to
bed. Sleep didn’t come readily, for there was to-day’s examination to
worry about, and to-morrow’s, too, for he hadn’t made much of that
two hours of preparation, he feared; and then there was this silly
quarrel with Laurie. He guessed he had been as much to blame as
his brother, but there was no sense in any one’s getting mad the way
Laurie had. When Laurie was ready to make friends, why, he’d be
ready, too, but that silly goop needn’t expect him to lick his shoes!
No, sir, if Laurie wanted to make up he could jolly well say so!
Sleep did come at last, and when he awoke it seemed hours later.
The room was in black darkness, but the squares of the wide open
windows were slightly grayer. What had awakened him he at first
didn’t know. Then his gaze caught a darker something against the
gray-black of the nearer casement opening, something that scuffled
on the stone ledge and grew larger as he wondered and watched.
He opened his mouth to speak, and then remembered that he and
Laurie were at outs. The form disappeared from sight, and footsteps
went softly across the boards, were muffled on the rug, and sounded
again by the door. The door was opened, and for a moment Ned
mentally pictured the boy peering anxiously out into the dim hall.
Then the door closed again, and after a short silence Laurie’s bed
creaked. To prove to the other that his return had not been made
unknown, Ned sat up in the blackness and thumped his pillow,
striving to express disapprobation in the thumps. Across the room
the faint stirrings ceased, and silence reigned again.
Ned smiled grimly. Laurie had probably thought that by being so
quiet he could get in without his brother’s knowing it, but he had
shown him! Then Ned’s satisfaction faded. What the dickens had
Laurie been doing out at this time of night? It must be twelve, or
even later! If he had been up to mischief—but of course he had; a
fellow didn’t climb into his room by the window unless he had
something to hide. Even being out after ten o’clock was a punishable
offense! Ned began to worry. Suppose some one had seen Laurie.
Why had Laurie gone to the door and listened unless he had
suspected some one of having seen him? The idiot! The chump! The
—
Over his head he heard a board creak. He listened. The sound
reached him again. In Elk Thurston’s room some one was up, too. Or
had he imagined it? All was quiet now. Was it possible that Laurie
and Elk had been settling their score? Surely not at this time of night.
And yet— From across the room came the unmistakable sounds of
deep and regular breathing. Laurie was asleep beyond a doubt! Ned
frowned disgustedly. Here he was worrying himself about a silly coot
that was fast asleep! He poked his head resolutely into his pillow. All
right! He guessed he could do that, too! And presently he did.
In the morning Ned waited for Laurie to break the ice, but Laurie
didn’t. Laurie went about his task of dressing in silence. There was a
sort of stern look in his face in place of the sullen expression of last
evening, and more than once Ned caught him looking across in an
oddly speculative way. The last time Ned caught him at it he began
to feel uneasy, and he wanted very much to ask what Laurie meant
by it. It was almost as if Laurie had caught him at something, instead
of its being just the other way about! But he was too stubborn to
speak first, and they went out of the room with the silence still
unbroken.
At breakfast, Mr. Brock, at whose table they sat, made the
disquieting announcement that Edward and Laurence Turner were
wanted at the Doctor’s study at 8:30. Involuntarily the gaze of the
two boys met swiftly. Each thought at once of examinations, although
further consideration told them that it was still too soon for any
shortcomings of theirs to reach the principal.
Although they had entered the dining-hall separately, now a
common uneasiness took them together to the Doctor’s, albeit in
silence. They were asked to be seated, which they accepted as a
favorable sign, but there was, nevertheless, something
unsympathetic in Dr. Hillman’s countenance. The latter swung
himself around in his chair and faced them, his head thrust forward a
little because of a near-sightedness not wholly corrected by his
spectacles. And then Laurie observed that the Doctor was gazing
intently at a point just under his left eye, and told himself that the
summons was explained. He was, though, still wondering why Ned
had been included in the party when the Doctor spoke.
“Laurence,” he asked, “how did you come by that contusion?”
Laurie hesitated, then answered, “I was having a—a little bout with
one of the fellows and he struck me, sir.”
“Who was the boy?”
“Thurston, sir.”
“Have you witnesses to prove that?”
“Yes, sir, several fellows were there. Pat—I mean Patton Browne,
and Proudtree and—”
“When did it take place, this—ah—bout?”
“Yesterday afternoon, about half-past five.”
The Doctor mused a minute. Then, “Which of you boys entered
your room by the window last night at about a quarter before twelve
o’clock?” he asked. The question was so unexpected that Laurie’s
mouth fell open widely. Then, as neither boy answered, the Doctor
continued: “Was it you, Laurence?”
“N-no, sir!” blurted Laurie.
Then, ere the words were well out, he wished them back, and in a
sudden panic he added, “I mean—”
But the Doctor had turned to Ned. “Was it you, Edward?” he
asked.
Ned’s gaze dropped from the Doctor’s, and for an instant he made
no reply. Then he raised his eyes again, and, “I’d rather not say, sir,”
he announced respectfully but firmly.
There followed another brief silence. Laurie was trying hard not to
look at Ned. The Doctor was thoughtfully rolling a pencil across the
big blotter under the palm of one hand. Ned watched him and
waited. Then the Doctor looked up again.
“You are, of course,” he said not unkindly, “privileged to refuse to
answer, Edward, but when you do there is but one construction to be
placed on your refusal. I presume that you did climb into your room
by a window last night. I confess that I don’t understand it, for this is
the first time since you came to us that your conduct has been
questioned. If you are shielding another—” his glance swept to
Laurie and away again—“you are doing wrong. Punishment that falls
on an innocent party fails of its purpose. I am, therefore, going to ask
you to reconsider, Edward. It will be better for every one if you
answer ‘yes’ or ‘no’ to my question.”
Ned returned the principal’s gaze straightly. “I’d rather not, sir,” he
replied.
“Very well, but I warn you that your offense is a very serious one
and that it calls for a drastic penalty. Were you alone in the—ah—
escapade?”
Ned looked puzzled. “Sir?” he asked.
“I asked you—But you need not answer that. I’ll put it another way.
There were two of you in the car according to an eye-witness. Who
was the other boy?”
“Car?” faltered Ned. “What car, sir?”
The Doctor frowned disapprovingly. “It is so futile, my boy,” he
said, “to act this way.” He turned to Laurie. “What do you know about
this, Laurence? You have said that you did not enter your room last
night by the window. At what time did you return to your room?
Where were you, for instance, at, say, a quarter to twelve?”
“I was in bed, sir.”
“What time did you go to bed?”
“About ten minutes past ten.”
“Where was Edward then?”
“In bed, sir, and asleep.”
“What? You are telling me the truth? Did you see him there?”
“Yes, sir.”
The Doctor frowned perplexedly. “Then you know nothing of any
one’s having entered your room by a window close to midnight?”
Laurie hesitated now. Then, “I went to sleep about ten minutes
after I got in bed, sir, and so I wouldn’t be likely—”
“Please answer my question,” interrupted the Doctor coldly.
“I’d rather not, sir,” said Laurie.
“One more question, then,” announced the inquisitor grimly. “Were
you in Mr. Wells’s automobile last evening when it collided with a
hydrant on Washington Street at approximately half-past eleven?”
“Why, no, sir! I didn’t know it had—had collided!”
Ned was looking rather white.
“You know nothing about the incident?”
“No, sir!”
“And you, Edward?”
“No, sir.”
“But, if you deny the automobile part of it, why not deny the rest? I
see, though. You knew that Mr. Cornish had seen you climbing in at
the window. I’m afraid you won’t get anywhere that way, Edward. Mr.
Wells’s car was taken from the front of the school last evening and
driven out Washington Street six blocks, where it was in collision with
a hydrant. It was abandoned there. A reliable witness states
positively that there were two persons in the car just before the
accident. About ten or twelve minutes later Mr. Cornish saw some
one climb up the Washington Street side of East Hall and disappear
through your window. Those are the facts, Edward. The evidence
against you is so far circumstantial, but you must acknowledge that
the incident of the car and that of your—of some one’s entrance into
your room by the window look to be more than a mere coincidence.
In other words, whoever entered your room at midnight was in the
stolen car a quarter of an hour before. That’s a fair and very natural
assumption. If I were you, I’d think the matter over carefully and see
me again before eight o’clock this evening, at which time it will come
before the faculty conference. And now, Laurence, let me have those
names once more.” He drew a scratch-pad to him and poised a
pencil. “You say Elkins Thurston struck you and that Proudtree,
Browne, and—who else was there?”
“Lew Cooper and Gordon Simkins were there when—right
afterward, sir, and I guess they saw it.”
“Thank you. That is all, then. I shall have to ask both of you to
remain in bounds until this matter is—ah—settled. Good morning.”
“But—but, Doctor, I’m—I’m on the baseball team, sir!” exclaimed
Laurie in almost horrified accents. “We play this afternoon!”
“I’m sorry, Laurence,” was the reply, “but until you are more frank
in your answers I shall have to consider you under suspicion, also.”
“Well,” said Laurie bitterly, when they were outside, “you certainly
have made a mess of things!”
“I!” exclaimed Ned incredulously, “I’ve made a mess of things?
What about you?”
“Me? What could I say?” countered Laurie hotly. “I did all I could!”
“All right,” said Ned wearily. “Let’s drop it. He won’t be able to pin
anything on you. You’ll get out of it all right.”
There was a trace of bitterness in Ned’s voice, and Laurie
scowled. “Well, he asked me so suddenly,” he muttered
apologetically, “I—I just said what came into my head. I’m sorry. I’d
have refused to answer if he hadn’t sprung it so quick.”
“It would have been rather more—rather less contemptible,”
answered Ned coldly.
Laurie flushed. “Thanks! I guess that’ll be about all from you, Ned.
When I want any more of your brotherly remarks I’ll let you know!”
He swung aside and left Ned to go on alone to No. 16.
The story of the purloining of the physical director’s blue roadster
was all over school by that time. Ned got the full details from Kewpie.
Mr. Wells had left the car in front of School Hall, as he very often did,
and was playing a game of chess with Mr. Pennington. Shortly after
half-past eleven he had looked for the car, had failed to find it, and
had hurried to the corner. There he had met a man coming down
Walnut Street who, when questioned, said that he had seen such a
car as Mr. Wells’s about five blocks east, where Washington and
Walnut Streets come together, not longer ago than five minutes.
There were two persons in it, and the car was not being driven more
than, possibly, twenty miles an hour. Mr. Wells had gone out Walnut
Street and found the car with one front wheel on the sidewalk, the
mud-guard on that side torn off, and the radiator stove in. There was
no one about. The car wasn’t very badly damaged, it was said, but
Mr. Wells was awfully mad about it. It was down in Plummer’s
Garage, and Ned could see it if he wanted to. Kewpie had seen it. It
looked fierce, but maybe it wouldn’t cost more than a hundred dollars
to fix it up again!
“Know who did it?” asked Ned.
“Me? I’ll say I don’t!” Kewpie laughed relievedly. “I guess it was
professional automobile thieves, all right, though. They were
probably heading for Windsor. That’s a dark corner up there, and I
guess they lost the road and turned too quick. They must have lost
their nerve, for Mr. Wells drove the car down to the garage and it
went all right, they say. Guess they thought it was done for and didn’t
try to see if it would still go. Sort of a joke on them, wasn’t it?”
“I suppose,” said Ned carelessly, “none of our fellows are
suspected?”
“Of course not. Why, it happened after half-past eleven! Say, you
haven’t—haven’t heard anything?” Kewpie’s eyes grew round with
excitement. “Say, Ned, what is it?” But Ned shook his head wearily.
“I know no more of the business than you do, Kewpie. Now beat it,
will you? I’ve got an exam at ten.”
CHAPTER XXIII
SUSPENDED
N ed didn’t get much studying done, though. Instead, he spent

most of the half-hour remaining before the examination in trying
to solve the mystery of the stolen car and Laurie’s part in the affair. It
wasn’t like Laurie to indulge in a prank so mischievous, and he could
scarcely believe that Laurie had taken part in the escapade. Still, he
had the evidence of his own senses. He had seen Laurie enter by
the window; and, too, he recalled the latter’s stated desire to drive
Mr. Wells’s car. At home in California Laurie was forever begging the
wheel away from his father and was never happier than when
steering the big car along the smooth roads about Santa Lucia. But,
if Laurie had taken Mr. Wells’s roadster, who had been with him? He
wished that Laurie hadn’t told a lie to the Doctor. That, too, was
something very unlike Laurie. Of course, as he had said afterward,
the question had been sudden and unexpected, and he had said the
first thing that came into his mind, but that didn’t excuse the lie.
Ned’s refusal to answer had been made in the effort to shift
suspicion from Laurie to himself, but he wondered now if it would not
have been as well to tell the truth. His self-sacrifice hadn’t helped his
brother much, after all, for Laurie was still suspected of complicity.
The affair would probably end in the suspension of them both,
perhaps in their expulsion. It was all a sorry mess, and Ned hadn’t
discovered any solution of it when ten o’clock came.
Rather to his surprise, he got through the examination, which
lasted until past twelve, very well. Then came dinner, at which
neither he nor Laurie displayed much of the exuberant spirit that
possessed their table companions. After the meal Ned went over to
the library for an hour. When he returned to No. 16 he found Laurie
standing at the window that looked southward toward the distant
ball-field, dejection in the droop of his shoulders. Ned felt very sorry
for the other just then, and he tried to find something to say but
couldn’t, though he cleared his throat twice and got as far as “Hm!”
You couldn’t see much of the baseball game from that window. The
diamond was at the far end of the field, and a corner of the football
stand hid most of it. Laurie found a book and read, and Ned began a
letter to his father. Somehow the afternoon wore away.
Kewpie burst in at a little before five, at once triumphant and
downcast. Hillman’s had won, 11 to 8, but Kewpie Proudtree had not
been allowed to pitch for even a part of an inning, and so his last
chance was gone, and if Pinky called that doing the square thing—
But Laurie broke in just then. “Can it,” he said gruffly. “You saw the
game, anyhow, and that’s more than I did!”
“That’s right,” said Kewpie, apologetically. “It’s a rotten shame,
Nod. What’s Johnny got on you, anyhow? You can tell me. I won’t
say a word.”
“He hasn’t got anything on me,” growled Laurie. “He just thinks he
has. Who pitched?”
“George started, but they got to him in the fourth—no, fifth, and
Nate finished out. Gee, they were three runs ahead of us in the
seventh!”
“Did Elk get in?”
“No, he’s got a sprained wrist or something. Pinky had Simpson, of
the scrubs, catch the last of the ninth. He dropped everything that
reached his hands, though.”
“Elk’s got a sprained wrist, you say? How’d he do it?”
“I don’t know. Maybe it isn’t a wrist. He’s got something wrong,
though, for I heard Dave Brewster talking about it.” After a minute
Kewpie returned to his grievance, and, since Laurie appeared busy
with his own thoughts, he was allowed to unburden himself to his
heart’s content. Ned condoled with him somewhat abstractedly.
When he had taken himself out Laurie broke the silence.
“With Elk out of the game,” he said bitterly, “I’d have had my
chance to-day, and then this had to happen!”
Ned might have reminded Laurie that he had only himself to
blame, but he didn’t. He only said, “I’m sorry, old son.” There was
sincerity in his tone, and Laurie heard it. He made no answer,
however. But later, at supper, their feud was dead, and after supper,
in the room, they talked enough to make up for twenty-four hours of
silence. One subject, though, was not mentioned.
Sunday morning the blow fell. There was another visit to Dr.
Hillman’s study. Both boys were again questioned, but their answers
did not vary from those they had given on Saturday. The Doctor
showed genuine regret when he made known the decision of the
faculty. Laurie had been exonerated from lack of evidence against
him, although it was apparent that the Doctor considered him as
deserving of punishment as Ned. Ned was suspended. That meant
that he would not be passed in his examinations and would have to
return next year as a lower-middler again. He might, as the Doctor
reminded him, study during the summer and so make the upper-
middle class during the fall term, however. As the present term was
so nearly at an end, the Doctor continued, Ned would be permitted to
remain at school until Laurie was ready to accompany him home.
The Doctor ended the interview with the suggestion that it would be
a manly act on the part of the twins to reimburse Mr. Wells for the
damage done to his car. Ned opened his mouth as though to say
something then, but he changed his mind and closed it again very
tightly. A minute later they were outside.
“Gosh, Ned, I’m sorry!” said Laurie miserably.
Ned nodded. “Thanks. It’s all right. One of us had to get it.”
“One of us?” repeated Laurie a bit blankly. “Why, yes, I suppose
so, but—”
“Well, you’ve got your baseball to look after, and I haven’t
anything. So it’s better they picked on me, isn’t it?”
“We—ell,” began Laurie. Then he stopped and shook his head in a
puzzled way. Finally, “You’ll stick around until Thursday, won’t you?”
he asked anxiously.
The other nodded. “Might as well,” he said. “I could get out now
and wait for you in New York, but I don’t see any reason why I should
spend all that money just to act haughty.”
The blow having fallen, Ned, who had already discounted it,
cheered up quite remarkably. After all, he told himself, he had saved
Laurie, and last autumn Laurie had saved him from something very
close to disgrace, and so this sacrifice only somewhat evened
accounts. He allowed himself to be persuaded to accompany the
others on the Sunday afternoon walk, only pledging Laurie to say
nothing of his suspension. It was not until Monday noon that the
news leaked out, and not until hours after that that the school began
to connect the incident of the wrecked automobile with Ned’s fate.
Even then most of those who knew Ned intimately refused to believe
that there could be any connection between the two things.
Questioned, Ned was very uncommunicative, and by Tuesday even
his closest friends began to waver in their faith.
Laurie went back to the baseball fold on Monday. Kewpie’s report
about Elk was true. Elk was nursing a lame wrist. He had, it seemed,
hurt it in wrestling with his room-mate. It had kept him out of the
game Saturday, and it prevented his doing any catching on Monday;
but on Tuesday the injured wrist appeared as good as ever, and
Laurie, who had been temporarily elevated to the position of first
substitute catcher, again dropped into third place. The Farview game
was due on Wednesday, which was likewise Class day and the final
day of the school term. On Monday Coach Mulford was very easy
with the first-string players but gave the substitutes a hard
afternoon’s work. Laurie caught four of the five innings that the
substitutes played against the scrub team. In the final inning he gave
place to Simkins and took that youth’s berth at first base. Tuesday
saw the whole squad hard at work in the final preparation for the
enemy, and no player, from Captain Dave Brewster down to the least
of the substitutes, had a minute’s respite. “You fellows can rest all
you want to after to-morrow,” said the coach. “You can spend all
summer resting if you like. To-day you’re going to work and work
hard.” Even Kewpie, who knew that Fate held nothing for him, was
subjected to almost cruel exertion. He pitched to Laurie until his arm
almost rebelled, and he was made to “dummy pitch” from the mound
and then field the balls that Pinky batted at him and to all sides of
him. And he ran bases, too, and Kewpie considered that the final
indignity and privately thought that the least Pinky could do was to
leave him in peace to his sorrow. But before Tuesday’s practice
began other things of more importance to our story happened. While
dressing Tuesday morning Laurie let fall a remark that led to the
clearing away of mistakes and misconceptions.
“You must have gone to bed with your clothes on the other night,”
he observed. “If you didn’t, you sure made a record!”
Ned stared. “What other night?” he asked.
Laurie floundered. Neither of them had referred to the matter since
Sunday. “Why—well, you know. The night you got in the window,”
Laurie explained apologetically.
“The night I got in the window! Are you crazy?”
“Oh, well,” muttered Laurie, “all right. I didn’t mean to make you
huffy.”
He went on with his dressing, but Ned still stared at him. After a
minute Ned asked: “Look here, old son, what made you say that?
About me getting in the window, I mean.”
“Why, nothing.” Laurie wanted peace in the family. “Nothing at all.”
“You had some reason,” Ned persisted, “so out with it.”
“Well, you were so blamed quick, Ned. You went to the door and
then I heard you get into bed about thirty seconds afterward. It don’t
seem to me that you had time to undress.”
“Let’s get this right,” said Ned with what was evidently forced calm.
“Sit down there a minute, Laurie. Why do you say it was I who came
through the window?”
It was Laurie’s turn to stare. “Why, why because I saw you! I
waked up just as your head came over the sill, you chump!”
“You saw my head come— Look here, are you in earnest or just
trying to be funny?”
“Seems to me it’s you who are acting the silly ass,” answered
Laurie aggrievedly. “What’s the big idea, anyway?”
“But—but, great Scott, Laurie,” exclaimed Ned excitedly. “I saw
you come in the window!”
“Cut the comedy,” grinned Laurie. “I wasn’t out, and you know it.”
“Well, was I, you poor fish? Wasn’t I in bed and asleep when you
came in, as you told Johnny you did?”
“Sure, but— Say, do you mean to tell me I didn’t see—”
“Of course you didn’t! But—”
“Then who did I see?” asked Laurie a trifle wildly.
“Who did I see?” countered Ned. “You say it wasn’t you—”
“Me! Hang it, I went to bed at ten and wasn’t awake again until I
heard a noise and saw you—well some one coming in that window!
Look here, if it wasn’t you, why didn’t you tell Johnny so?”
“Because I thought it was you, you poor prune!”
“What! But I’d said—”
“Sure you had, but I’d seen you with my own eyes, hadn’t I?”
Laurie shook his head weakly. “This is too much for me,” he
sighed. “It wasn’t you and it wasn’t me but it was one of us! I pass!”
“But it wasn’t one of us,” exclaimed Ned. “That’s what I’m getting
at. Don’t you see what happened?” Laurie shook his head.
“Listen, then. We were both asleep, and we each heard the noise
and woke up. Some one came through the window, crossed the
room, opened the door, looked out to see that the coast was clear,
went out, and closed the door after him.”
“But I heard you get into bed!”
“No, you didn’t. You heard me sit up and punch my pillow. I wanted
you to know that you weren’t getting away with it. For that matter I
heard your bed creak and thought you were getting into it.”
“I sat up, too,” said Laurie. “Gee, that’s a queer one! All this time I
thought it was you and could have kicked myself around the block for
yelling ‘No!’ when Johnny asked me that question! Then—then who
the dickens was it, Ned?”
“That,” answered Ned grimly, “is what we’ve got to find out. Just
now it’s up to us to get out of here before we miss our breakfasts!”
“Hang breakfast!” shouted Laurie. “This is better than a hundred
breakfasts! Why—why, it means that you—that you aren’t
suspended! It means—”
“Put your collar on, and make it snappy,” laughed Ned. “We’ve got
some work ahead of us this morning!”
After breakfast they hurried back to No. 16, barred the door
against intruders, especially Kewpie, sat down at opposite sides of
the study table, and faced the problem. They continued to face it
until nearly eleven. They examined the window-sill for clues, and
found none. They leaned out and studied the ivy by means of which
the mysterious visitor had reached the second story, and it told them
nothing, or so it seemed at the moment. As they turned back to the
room Ned said idly: “It’s lucky the fellow didn’t have to get to the third
floor, for I don’t believe he could have made it. That ivy sort of peters
out above our window.”
Laurie nodded uninterestedly and silence ensued, just as silence
had ensued so frequently before in the course of morning. Then,
several minutes later, Ned said suddenly, questioningly:
“Thurston!”
Laurie shook his head. “Not likely. Besides, what reason—”
“Wait a minute. I didn’t tell you. It didn’t seem important. After I’d
settled down again that night I heard the floor up-stairs creak twice. I
wasn’t just certain then, but now I am! Elk Thurston was moving
about up there, Laurie!”
“Well, what if he was? That doesn’t prove—” He stopped and
frowned intently. “Hold on, though, Ned! What about Elk’s wrist?”
“We’ve got it!” cried Ned.
“Yes, maybe. Let’s go slow, though. You don’t happen to know
whether Elk can drive a car, do you?”
“No, but I’ll bet you anything you like that he tried to drive that one!
Look here, our window was open and it was easy to reach. He
couldn’t have made his own without chancing a fall. He trusted to our
being asleep. He—”
“What about the other fellow, though?” asked Laurie. “We didn’t
see—”
“No, but maybe he got in first. Maybe it was really he who awoke
us. Come to think of it, you said that when you woke up the fellow’s
head was just coming into sight. Well, in that case there wouldn’t
have been enough noise—”
“By jiminy, that’s so! Bet you that’s what happened. But who—
Say, maybe the other fellow was Jim Hallock!”
“Just what I was thinking,” agreed Ned. “I don’t see, though, how
we can prove anything against either of them. Look here, son, I
guess the best thing we can do is see Johnny and tell him all about
it. After that it will be up to the faculty. Come on!”
They had to wait some time for an audience, but finally they were
facing the Doctor, and Ned, as spokesman, was saying very
earnestly: “Neither Laurie nor I was out of our room after ten o’clock
Friday night, sir. Somebody did come in our window, though, and
woke us up. I thought it was Laurie and he thought it was me, and
that’s why I didn’t want to answer your question, sir.”
Now, nothing could have been clearer and simpler than that and
yet, when Ned had finished, the principal blinked behind his
spectacles, gazed a moment in silence, and then waved a hand.
“Sit down, boys,” he said. “Now, Edward I think you’d better say
that all over again.”
CHAPTER XXIV
MR. GOUPIL CALLS
A fter practice that afternoon Laurie returned to the room to find

Ned engaged in sorting things out preparatory to packing up.
When Laurie entered, however, the other paused in his effort to stuff
more rubbish into an already overloaded waste-basket and
announced in triumph, “We had it right, partner!”
“Elk Thurston?”
“Elk and Jim Hallock. Elk’s just left here.”
“Left here? You mean he was in to see you?”
Ned nodded. “Yes. It was rather decent of him, I think. Take that
idiotic expression from your face and sit down. This is how Elk tells
it. He and Jim were looking out of their window that night and saw
the lights of Mr. Wells’s car on the other side of the hedge. One of
them said something about Mr. Wells always leaving his car around
and what a joke it would be if it wasn’t there when he came back for
it. Well, that idea sort of stuck, and after a while Elk suggested that
they sneak down and run the car off around the corner. Elk says that
Jim usually wouldn’t have gone in for anything like that on a bet, but
there’d been some tough exams that day, and Jim was sort of keyed
up. Anyhow, they sneaked down-stairs after a while and got out by
one of the windows in the recreation-room. They didn’t dare try the
front way, for Cornish had his study door open. They put the brakes
off and tried to push the car toward Washington Street, but it was
heavy, and after they’d got it a little ways they decided to start it and
run it around the corner. So they did, pretty sure that it was too far off
for Mr. Wells to hear. Elk took the wheel and they went to
Washington Street. Then, he says, the thing was working so pretty
they thought they’d go on further. When they got to where
Washington joins Walnut it was pretty dark, and he swung to the
right too soon.
“That’s when they hit the hydrant. Of course, they were scared
pink, and Elk shut the motor off and they beat it as fast as they
could. When they got back here they found that some one had been
prowling around and had locked the window. Then they saw our
windows open and decided to climb up by the ivy. Elk says they
hoped we’d be asleep. If we waked up they meant to tell us and ask
us to keep mum. Jim climbed up first and made it all right, but Elk
had hurt his wrist when the car struck the hydrant, and he had a hard
time of it. They didn’t either of them know that Cornish had seen
them. For that matter, he only saw one, I guess, and that one was
probably Elk, for he says it took him two or three minutes to get to
the window because his wrist hurt him so. Seems that Jim left the
hall door open after him, but the draft closed it, and that’s what woke
us up, I guess. Well, what Elk came for was to say that neither of
them knew they’d been seen and that they hadn’t meant to throw
suspicion on us. He says if they’d known that Cornish was prowling
around they wouldn’t have entered our window. He was very
particular about making that clear. Guess he thought you might think
he had done it on purpose to get even with you. And that’s that, old
son.”
Laurie nodded thoughtfully. “Kind of too bad,” he mused. “I
suppose they didn’t intend anything but a sort of joke on Mr. Wells.
Did he tell you what they were going to get?”
“Get? Oh, they’re suspended, he says. He seemed to feel worse
about Jim than about himself. Do you know, old son, after all Elk isn’t
such a bad sort. At least, that’s the way it strikes me after hearing his
spiel. He says he’s not coming back next year. He’s going to tutor
this summer and try and make college in the fall.”
“Yeah,” said Laurie abstractedly. “Well, I’m sort of sorry for him.
And of course he didn’t mean to get us in wrong.” He lapsed into
silence. Then, abruptly, “Cas Bennett split his finger with a foul tip
about half an hour ago,” he announced.
“He did?” exclaimed Ned. “Gosh, that’s tough luck! Will it keep him
out of the game?”
“Yes,” replied Laurie.
“That is tough! Say, what are you looking so queer about?”
“Just thinking,” answered Laurie. “You try it.”
“Huh?”
“Use the old bean, son. Cas has split his finger, Elk’s suspended
—”
“Great jumpin’ Jehoshaphat! Why, then, you—you—”
“Correct,” said Laurie. “I’ll have to catch to-morrow, and—and at
the present moment, Ned, I’m scared to death!”
That had been a day of events, and it was not yet over. Attic
Society was giving its usual end-of-the-term blow-out that evening,
and both Ned and Laurie were invited. The affair began at eight, and
at half-past seven they were in No. 16 putting the finishing touches
to their toilets. Although it was a stag-party it called for best clothes
and polished shoes and carefully brushed hair, and Laurie was trying
hard to subdue a rebellious lock on the crown of his head when there
came a knock on the door. Both boys shouted “Come in!”
simultaneously. Then the door was opened, revealing Mr. Cornish,
the hall master, and a stranger. The boys grabbed for their coats,
Laurie dropping a military brush to the floor with a disconcerting
noise. Mr. Cornish ushered the stranger in but himself came no
further than the door-sill.
“Here is a gentleman to see you, Laurence,” said the instructor. “I
was quite certain you were in, and so I brought him up.”
Mr. Cornish smiled, nodded to the guest, who bowed impressively,
and departed, closing the door behind him.
“Very glad indeed—” began Laurie.
“Have a seat, won’t—” supplemented Ned.
“Thank you.” The stranger again bowed and seated himself,
placing a cane across his immaculately clad legs and balancing a
somewhat square derby hat perilously atop. “I begin by offering you
my apologies for this intrusion,” he continued.
“Not necessary,” mumbled Laurie, his gaze busy with the guest.
The latter appeared to be about fifty, was under rather than over
average height, and was very broad and thick and, like his derby,
rather square of contour. He even had a distinctly square face which
began very high up, because of the disappearance of what hair may
have adorned the front of his head at one time, and ended in an
auxiliary chin. He wore a very black mustache whose ends were
waxed to sharp points. His eyes were quite as black and almost as
sharp as his mustache. He looked foreign, and, indeed spoke with
more than a trace of accent, but he was evidently a gentleman, and
he impressed the boys very favorably.
“With your permission,” he continued, “I will introduce myself.” He
regarded Laurie. “I have the honor of addressing Mr. Laurie Turner?”
Laurie nodded. The guest carefully secured hat and stick, arose, and
bowed deeply. “I,” he announced then, “am Mr. Goupil.”
For an instant silence ensued. Then, “Mister—I beg your pardon,”
said Laurie, “but did you say Goupil?”
“Goupil,” confirmed the gentleman, bowing again and smiling very
nicely.
“You mean,” stammered Laurie, “the Mr. Goupil? Of Sioux City?
Miss Comfort’s Mr. Goupil?”
“Surely.”
“Why—why, then,” exclaimed Laurie, “I’m mighty glad to meet you,
sir.” He stepped forward with outstretched hand, and Mr. Goupil
enfolded it in a far more capacious one. “And this is my brother Ned.”
Mr. Goupil then shook hands with the amazed Ned. After that they all
sat down. Mr. Goupil arranged stick and hat with precision, cleared
his throat, and began:
“My dear sister-in-law has told me of your most kind efforts in her
behalf, and I have presented myself to make explanation and to add

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Molybdenum and Tungsten Enzymes -

Bioinorganic Chemistry 1st Edition

Molybdenum and Tungsten Enzymes - Biochemistry 1st

Molybdenum and Tungsten Enzymes - Spectroscopic and

Chemistry of metalloproteins problems and solutions in

Insights from imaging in bioinorganic chemistry First

Applications of Density Functional Theory to Biological

Metallocofactors that Activate Small Molecules With

Nighthawking 1st Edition Russ Thomas [Thomas

Mixing Music 1st Edition Russ Hepworth-Sawyer

RSC Metallobiology Series

Titles in the Series:

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Molybdenum and Tungsten

RSC Metallobiology Series No. 6

Print ISBN: 978-1-78262-877-4

© The Royal Society of Chemistry 2017

All rights reserved

Published by The Royal Society of Chemistry,

Registered Charity Number 207890

For further information see our website at www.rsc.org

RSC Metallobiology Series No. 6

a wide range of metabolic pathways and play particularly prominent roles in

RSC Metallobiology Series No. 6

and molybdenum complexes when, beginning in the late 1970s, he became

Chapter 1 An Overview of the Synthetic Strategies, Reaction

Introduction and Overview  1

Chapter 2 Pterin-Inspired Model Compounds of

RSC Metallobiology Series No. 6

3.1 I ntroduction to Molybdenum and Tungsten  68

Chapter 4 Comparative Kinetics of Enzymes and Models  94

4.1 A ctive Sites of Molybdenum and Tungsten

Chapter 5 Synthetic Models of the Nitrogenase Clusters  130

5.1 I ntroduction  130

5.6 Cluster Assembly Mechanisms:

Chapter 6 Synthesis of Mono- and Bisdithiolene Molybdenum and

6.1 I ntroduction  166

Chapter 7 Models for the Xanthine Oxidase Family of Enzymes  194

7.1 I ntroduction  194

Subject Index  239

An Overview of the Synthetic

Introduction and Overview

RSC Metallobiology Series No. 6

An Overview of the Synthetic Strategies, Reaction Mechanisms 3

lacking an interstitial atom, now recognized to be carbon.

Semisynthetic enzymes will have to be prepared and compared for their

An Overview of the Synthetic Strategies, Reaction Mechanisms 5

chemistry per se is well understood, although elaborating pterins with pyrane

to acetaldehyde relevant for the extraordinary tungsten-dependent acetylene

An Overview of the Synthetic Strategies, Reaction Mechanisms 7

I am thankful to all of them for having expertly detailed the fundamental

RSC Metallobiology Series No. 6

2.2 Unveiling the Pterin Component of Moco

2.2.1 The Pterin is Discovered

Scheme 2.1 The results of redox titration experiments on molybdoenzymes.

Santamaria-Araujo et al. proved that 3 was a pyranopterin in the reduced state

2.3 Development of Pterin-Inspired Models

2.3.1 First-Generation Models

2.3.1.1 Models Where Molybdenum is Coordinated Directly to

proposed by Rajagopalan, suggested several metal binding sites in addition

dimeric species [Mo2O5(xanthopterinate)2]2− (Figure 2.10) where, amusingly,

N ed didn’t get much studying done, though. Instead, he spent

Chapter 1 An Overview of the Synthetic Strategies, Reaction

Introduction and Overview 1

Chapter 2 Pterin-Inspired Model Compounds of

3.1 I ntroduction to Molybdenum and Tungsten 68

Chapter 4 Comparative Kinetics of Enzymes and Models 94

4.1 A ctive Sites of Molybdenum and Tungsten

Chapter 5 Synthetic Models of the Nitrogenase Clusters 130

5.1 I ntroduction 130

5.6 Cluster Assembly Mechanisms:

Chapter 6 Synthesis of Mono- and Bisdithiolene Molybdenum and

6.1 I ntroduction 166

Chapter 7 Models for the Xanthine Oxidase Family of Enzymes 194

7.1 I ntroduction 194

Subject Index 239

2.2 Unveiling the Pterin Component of Moco

2.2.1 The Pterin is Discovered

2.3 Development of Pterin-Inspired Models

2.3.1 First-Generation Models

2.3.1.1 Models Where Molybdenum is Coordinated Directly to