262
In some cases, one of the subumts a as
The aerobic respiratory chain of
Eschedchia co#
YasuhiroAnraku and Robert B. Gennis
The aerobtc respwatory chain of Eschenchla coh couples the ox~dauon o f orgarac substrates,
such as succmate, to the generatzon of a proton motwe force across the cytoplasnuc mem-
brane. The mechanmn by whtch thts Is accompbshed ~s quae thfferent than found m the
mztochondnal respiratory system, as predzcted by D Kedm I nearly 50 years ago Aerobtcally
grown E cohhasnocytochrome~ andnoequzvalenttothemuochondnalComplex lll (bcl
complex) or Complex IV (cytochrome c oxutase) Instead, two enzymes m the E coh cyto-
plasnuc membrane, the cytochrome o and O,tochrome d complexes, oxuhze ubulmnol and
drealy reduce molecular oxygen to water, c o n c o ~ genermv~ an elearochenucal
proton grachent across the membrane
The major functzon of the respiratory
chains of aerobically gro~n bactena is
the electrogemc translocatlon of protons
out of the cytoplasm The resulting elec-
trochenucal proton gradient across the
cytoplastmc membrane ~s then uUhzed
to dnve ATP synthesis, solute uptake,
and other energy-requmng, membrane-
associated processes such as flagellar
motion Before this decade, little was
known on a biochemical level about the
components of bactenal respiratory sys-
tems, but recent progress has been rapid
The pnmary motivation for these studies
is to elucidate the enzymology of proton
translocatlon across the membrane and,
for this purpose, bactena offer some
advantages over eukaryot¢ systems (1)
The number of subumts m the bactenal
enzymes is far less than in the mRo-
chondnal r e s w a t o r y enzymes, making
structure-function correlations more
feasible expenmentally. (2) it is relatwely
easy to apply techniques of molecular
genetics to bacterial systems, especially
E colt.
Bacterial respiratory chains
The main focus of this review is to
describe the current status of work on
the aerobic respiratory system of E colt
However, in order to apprec,ate better
the context of th~s work, some back-
ground ,n bacterial resp, ratory systems is
useful F~gure I shows a general scheme
wh,ch can be appl,ed to many bacter,a,
though certainly other var, at~ons exist
Bacteria generally have more than one
terminal ox,dase there are several
dehydrogenases which can feed elec-
trons Into the system, and two or three
terminal Oxldases which can each reduce
Y Anrakatsm th,"Departmentof Biology, Facultyof
Sctence UntverslB,of Tokyo Hongo, Tokyo 113.
Japan, R B Gennts L~ at the Department~ of
Btochermstry and Chemmry, Unaer~tB, of IIhnols,
505 South Mathew~ St, Urbana, IL 61801, USA
t~ 1987 El~,,qer P.bhgallOn~ Cdmbndgc (1376- lilh~%87/~12OIl
molecular oxygen to water Depending
on the growth conchuons, one of the
ox,dases may predominate For exam-
ple, in E coil, the cytochrome o term,nal
oxldase complex is predominant when
the cells are growu with high aeration,
and the cytochrome d complex is pre-
dominant when the oxygen tension is low
(see Ref 2)
Another feature to note is that some
bactenal ox~dases utlhze cytochrome c as
substrate, similar to the matochondnal
cytochrome c oxldase, others utilize
ublqumol as shbstrate, and, as such,
function as ublqumol oxldases The
bactenum Paracoccus denttrtficans, for
example, contains oxadases of both
types. The branch of the P. demtrlftcans
respiratory chain containing the cyto-
chrome c oxldase has been intenswely
studied because of Rs similarity to the
mltochondnal system (see Ref. 2)
Eschenchla cob does not contain a
cytochrome c-dependent branch of ~ts
respwatory chain However, it does con-
tam two oxldases which both oxidize
ublqumol Such duplication appears un-
necessary genetic studzes have shown
that the ehmmauon of enher does not
result in any growth deficiency under
laboratory condmons ~ 4 Presumably,
there as a selective advantage to having
both oxldases in nature, possibly related
to either the higher affinity for oxygen of
the cytochrome d complex, or the differ-
ence in sensnlvlty to respiratory
infubltors (see Ref 2) The bloenergeUc
effioency, that ,s protons translocated
per electron, seems to he the same
regardless of which oxldase is present
The bacterial oxldases are a diverse
collection of heine proteins, and heine a
and Cu 2+ are not reqmred prostheuc
groups as they are in the mltochondnal
analogue (Table I) E,lzymes in class !
are slmdar to the mrochondnal oxldase,
but contain only two or three subumts
TIBS 12 - July 1987
c-type cytochrome (caa 3 type) Class II
contains enzymes wluch contain proto-
heme IX (heine b) as a prosthetic group,
and which also function as cytochrome c
ox]dases these are termed o-type
oxldases Note that some enzymes In this
class are zsolated tightly bound to c-type
cytochrome subumts (co type) In most
cases, httle is known about the enzymes
m this class apart from the results of kine-
tic studaes and analysis of subumts by
SDS-PAGE
Classes Ill and IV are ublqulnol
oxldases, and the two ox]dases from E
coh constitute the primary examples that
have been characterized These bd and
o-type ublqulnol oxtdases exist m other
bactena 2,s but have not been examined
,n detail m these systems
Composition of the E. coh aerobic
respiratory system
All of the components of the respara-
tory system are Iocahzed in the inner
(cytoplasmic) membrane There are two
major groups of enzymes listed m Table
I! whach partlcapate m the system Group
! - those enzymes that oxldzze orgamc
substrates and reduce ubaqulnone, and
Group H - the two enzymes which
oxadaze ublqumol and reduce oxygen to
water
A macroanalyacal technique utd,zlng
high performance liquid chromatog-
raphy (HPLC) has been developed
which can be used to separate and quan-
ufy all of the cytochrome components of
SUCCINATE NADH D-LACTATE
',, I /
1
UBIQUINONES
°°m°'- I
| |a
I cytochromec ub,qulnol I
Lox,dos- I I ox,dos.sI
Ftg I A 8enencschemefor bactertalrespiratorysys-
tems One vanauon has an elearon transferchain
with componems eqt.valent to those m the
muochondna Cytochromec cames electronsfrom
the bg complex to be oxutase Bacteriaalso have
abtqumol oxMases whtch can bypass the OtO-
chrome c-dependent branch Often two or three
o:udase~mav be presenttn the bacterialmembrane
 "FIBS 1 2 - J u l y 1987
263
the E c o b membrane6 This method has Table I Baclenal terminal oxutases which have been p t ~ d
been useful in demoustratmg an addi-
tional cytochrome b561,whose function is Specms Prosthetic grouPSb Subumt
unknown, but wlach has now been well tool wt
(x 10-~)
characterized both biocbemmally and
genetically~.s C/as~! Cytochromec oxtdasescontam.ngheinea and Cu2"(aa3-,caa3-andat-types)c
I~.acoccus denanficans~ ~ berne a, Ca2 * 45.28
Ublqumone-8, the major ubtqumone
Bacdlussubalts~ e hemea, (Ca:+) 57,37,21
species present dunng aerobic growth Tiuobacdlusnovellus a e bemea,Cu2+ 32,23
has eight isoprene umts in its side chain Nitrobacteragdts~ e hemea Cu2+ 40.27
(see Ref 9) The ubtquinone in the Rhodobactersphaeroutese hemea, (Cuz+) 45,37,35
mltochondnon is ublqumone-10 In the NItrosomonaseuropaea r heinea, Cu2÷ 50,33
membranes of aerobically g r o w n E coh Thermuslhermophdusa e hemec. hemea Cu-'* 55,33
cells there are at least 50 molecules of Thennopl~cbacter, urn PS3~ ~ hemec, heinea, Cu2• 56,38.22
Bacdlusfirmus RABg hemec, hemea, (Cu2+ ) 56,30,14
ubtquinone per oxadase complex9 This
compares w~th a value of about seven for C/assH Cytochmmec omdasescontainingheineb (o- and co-types)
the mttochondnal systemm The reason Azotobacter vmelandu h hemeb 28
for the large excess ~s not clear, nor is ~t Rhodobactercapsulalm, hemeb 65
known whether this ~sregulated Rhodobacter splmerotdc~, heineb 23,|9,6
Pseudomonas aerugmosa, heinec, heineb 29,21,12,10
The enzymatic and molecular properties Rhodopseudomonaspalusms, hemec hemeb 30,26,13 10
Methylopbdus methylotrophm, heinec, heineb 29,21
of two terminal oxidases
Under normal aerobic laboratory Class III Qmnolo~adasescortmmng heineb and heined (chlonn)(bd-type)
growth conditions, both of the E coh Escherzchulcoha ! heineb. hemed 58,43
omdases are present m the cytoplasmic Photobactenwnphosphoreumk heineb, heined 54.41
membrane The genes encoding cyto-
chrome o (cyo) and cytochrome d (cyd) C/ass/V Omnolomdasescontammgheineb (o-type)
E,uhem.htacohaJ berneb, Cu 2" 66,36,18,13
complexes map at nunutes 10.2 and
16 7, respecnvely, on the E colt genetic "Shownm vitroto be directlyrevolvedmprotontmnslocauon
linkage map 3 4. Strains which are either bHemeb denotesprotohemeIX Heined is a chlonn The parenthesesdenote that Cu2. is assumedbut
c y o - or c y d - are fully capable of aerobm not actuallyshownto be presentm the punfiedenzymes
growth under all condmons examined, ,Earlierclass~mnonshadartype ox~dasesasa separategroup Theabsorptionpeak near595nmmarking
these cytochromes,m somecases,msuhsfroma componentm the bd-type complexesand m othercases
with no alteration m either the rate or ,s due to a variantof the aa3-typ¢enzyme
extent of growth Stratus lacking the 'tVerylowturooversuggeststMsmaynot o~od,zecyiochromec m vt~o
cytochrome d complex are, however, eLud~g. B (1987)FEMS Mtcrobtol Rev 46, 41-56
more sensitive to cyamde and azlde, a rDaspmto,A A, Ltpscomb,J D and Hooper,A B (lg86)J Bml Ckem 261,17048-17056
feature which has proved useful m gen- sgatada, M J and Krulmeh,TA (1984)./ Bactenol 158,963-966
etic selections Stratus lacking both hyang,T 0986) Bmclum B~ophys Aaa 848, 342-351
oxtdases ( c y o - c y d - ) roll not grow ~Forreferences,see Kranz.R G and Genres,R B {1985)J Bactenol 161,709-713
~Seetext
aerobically on non-fermentable sub- kKonisht,K, OuchJ,M, Kda. K and Honkosht,I (1986)J flmehem 99, 1227-1236
strates such as lactate or succmate4. Both
of these enzymes must catalyse the four-
electron reduction of molecular oxygen There are no substantml sequonce Antiboches directed
c h a r a c t e n , ed t5
and the two-electron omdatmn of homologms wRh other sequenced pro- against sub-rot I l~,, as well as controlled
ublqmnol-8 Enzymologlcal studies have terns Electrochemical and spectroscopic proteolysJs of this subumt within the
progressed using punfied preparations studies t4 have shown that this enzyme complex (Lorence and Gennts, unpub-
of both enzymesu, the pertinent proper. complex contains three distract cyto- hshed), specifically ehmmate the ability
tins of winch are summarized m Table chrome components and four heine of the enzyme to oxidize ubiqumol
II! Note that the cytochrome d complex prosthetic groups two protoheme IX These expenments suggest that cyto-
has the highest affimty for 0 2 of all the groups (cytochromes bsss and bsgs) and chrome bs5a ts directly revolved in
tenmnal omdases so far known, whereas two chlonns (cytochrome d) There is no ublqumol omdatton (see Table Ill)
it has the lowest affinity for KCN or Cu 2. or non-heine iron present tn the (2) Cytochrome boo~ (Era =
NaN 3 (Ref 11) enzymet112 The properties of the three + 1(30mV) The spectroscopic feature (a
T h e c y t o c h r o m e d c o m p l e x has been cytochrome components of the complex peak at 595 mm m the reduced-minus-
purified to homogeneltytl.t2 and con- can be summanzed as follows oxidized spectrum) associated with this
tams two subumts m a I 1 complex Sub- component of the complex has previ-
umt ! (mol wt 58000) and subumt 11 (I) C y t o c h r o m e bsss ( Era = + 180 mV) ously been ascnbed to cytochrome a t
(43000) are both encoded by the c y d has the spectroscopic properties typical (see Ref 2) However. the prosthetic
locus, whmh appears to be a two-clstron of b-type cytochromes 14 and has been group of this cytochrome Is not heme a,
operon3, t3 The c y d locus has been defimtivelv shown to be located on sub- but protoheme IX tz 14 The spectrum of
cloned ~3 and the DNA sequence deter- unit ! Although the two subumts of the this component was deduced by elec-
mined (Genres et a l , unpubhshed) The complex cannot be split apart without trochemmai manipulations and is similar
deduced prote,n sequences mdmate that denatunng the enzyme, geneUc mampu- to that of 5-coordinate (high spin)
both subunlts I and !I are transmem- lattons have allowed subumt ! to be syn- catalases and peromdases which contain
brane proteins, and each probably thesized in the absence ofsubumt ll, and protoheme IX j4 The enzyme, however,
crosses the membrane several hmes this subumt has been tso!ated and has no catalase or peroxtda~ acttwty,
264 TIBS 12-July 1987

TableII Enzymecomponents of the aerobic resptratoo, system of E coh whichha~e beenpunfieda

Components Prosthetic groupsb Subumt mol wt Comments~
(X l0 -3)

Group I Dehydrogenases
NADH dehydmgenase FAD 47 Cloned, sequenced, not transmembrane
Succmate dehydrogase FAD (covalently, linked), 64,26,14.13 Cloned, sequenced, contams cytochrome bsse as one of two small
non-heme non. heine b 'anchor' subumts to which the larger catalytic subumts me bound
D-Lactate dehydrogenate FAD 65 Cloned, sequenced
t-Lactate dehydmgenase FMN 43 Induced
Py~vate oxldased FAD 60 (teiramer) Cloned, sequenced, hpld-reqmnngperipheral membrane enzyme
sn-Glyceml-3-phosphate FAD 58(d;mer) Penpberal membrane enzyme
dehydrogenase (aerobic)
D-An'unoacid dehydrogenase FAD, non-heine iron 55,45 D-Alanlne is best subslrate
Prohne oxldased FAD 130 (dlmer) Cloned, penpheral membrane enzyme

Group il Temunal oxldase~

Cytochmmeo complex heme b. Cu 2• 66,36.18,13 Cloned, predominates at lagh aemuon
Cytochmmedcomplex heme b, heine d (chlonn) 58.4"; Cloned, sequenced, predominates at low aeratmn

M~cellaneous
Cytochrome b~l heine b 18 Cloned, sequenced
aData from Ref 30
bHeme b denotes protoheme IX. heme d is a chlonn
cpenpberal membrane enzyme denotes a protein that is easily dislodged from the membrane and can be isolated as a water-soluble spemes
dThese are dehydrogenases and not true oxldases since they do not directly interact with 0 ,

and this group does not appear to bind to
CO or to O 2
(3) Cytochrome d (Em -- + 2 6 0 m V )
There are two chlonn molecules present
in the enzyme 14 The structure of cyto-
chrome d has recently been elucidated
(Fig 2), and it appears to be denved
from protoheme IX by a reductwe
dlhydroxylatlon in pyrrole nng C (Ref
17). Optical and electron spin resonance
(ESR) studies clearly show that the
cytochron ~ d component of the complex
binds to 0 2 and CO, and is directly
involved in the activation of 0 2 and its
reduction to water (see Refs 14 and 18).
The redox r e a s o n catalysed by the
cytochrome d complex generates an
electrochemical proton gradient of 150-
180 mV (negative inside), across the
membrane, indicating that the complex
serves as a coupling site of oxldatwe
phosphorylatlonn,tg.20 The purified
cytochrome d ~omplex can be reconsti-
tuted m umlamellar phosphohpld vesi-
cles with an cnentatlon which is at least
Tablelll Enzyoumcpropernes of Otetwo ternunal~~adases
Cytochrome ocomplex
Substrate (Kin, pM)
Ublqumol-!
Menadlol
O2
inhibitor (mM)a
KCN
NaN3
H202
HQNO
I~enmchnA
~oncentration reqmred for 50% inhibition of ublqumol-I oxldase actlwly
75% right-side-out, that is, similar to the
onentaaon m the E coh membranO 92o
By prepanng vesicles incorporating
ublquinone-8 in the bdayer and mare-
taming this qumone in the reduced state
by using an excess of the flavoprotem
pyruvate oxtdase, it was shown that the
cytochrome d complex utilizes ublqui-
nol-8 as a substrate. The maximal turn-
over of the complex observed in this sys-
tem is about 150 e -1, which is faster than
the rate observed m either intact cells or
in membrane preparations Turnover of
the cytochrome d complex tn these vest-
des generates a voltage difference across
the bilayer of at least 180 mV (negative
inside) and results in the translocauon of
oae H + per electron from inside to out-
side, comparable to the values associated
with the respiratory chain in the E cob
membrane
The cytochrome o complex has also
been purified and proved to be a coup-
ling site for oxtdatlve phosphoryla-
uon 2t-26 The reconstltuuon results are
Cytochmme d complex
48 230
38 17
29 0 38
0 01 2 previously, the ESR and optical data
15 400
300 120
0 002 0 GO'/
0 002 0 015
very smnlar to those obtained w~th the
cytochrome d complex The maxamal
turnover m the reconsututed system is
over 500 e -t using ublqmnol-8 as a sub-
strate 24 Despite the apparent functional
equivalence, the structures of the two
oxtdases appear to be very different The
cytochrome 0 complex contains two to
four subunits by SDS-PAGE analysis 21-23.
There appear to be two protoheme IX
groups (cytochrome bss5 and b562) and
two Cu 2+ atoms per complex 22,23. The
two berne-two Cu 2÷ structure Is more
reminiscent of the mttochondrial oxldase
than the cytochrome d complex. The cvo
gene which encodes the cytochrome o
complex has been cloned and DNA
sequencing is m progress (Genres et al,
unpublished) Questmns concerning
potential sequence homologies will be
addressed m the near future
Biophysical studies of the cytochromes in
the oxidase complexes
The avatlablhty of the punfied
enzymes as well as membranes enriched
m either oxldase has facthtated btophysl-
f:al approaches, m particular ESR For
example, ESR signals from each of the
cytochrome components of the cyto-
chrome d complex have been assigned
(Ref 18 and Memhardt, Genres and
Ohmshl, unpubhshed) As mentioned
(Ref 27 and Lorence and Genres,
unpubhshed) show that the cytochrome
d component of the complex is bound to
molecular oxygen at room temperature
Furthermore, both ESR and optical data
TIBS 12-July 1987
suggest that the enzyme may pass
through a peroxy-mtermedtate bound to
cytochrome d dunng the catalyUc cycle
it Is also mdtcated by ESR expenments
that cytochromes b595 and d are physt-
cally close (Ref 18 and Anraku et al,
unpubhshed) The probable sequence of
electron flow ts as follows:
cyt bsss--+cyt b595---~cyt d---~O2
Quesuons concernmg the exact nature of
~he mtermedtates revolved m the two-
electron ox~datton of ublqmnol and four-
electron reduction of oxygen have yet to
be addressed.
The cytochrome o complex has also
been examined by both ESR ~s and res-
onance Raman spectroscopy2S The data
obtained are conssstent with a model that
has some SlnUlanty with the rmtochond-
hal aa~-type ox~dase2~ It ~s suggested2s
that the cytochrome b~2 component of
the complex ~s analagous to cytochrome
a and does not bind to CO or 02,
whereas the cytochrome b~s5component
ss analagous to the 02 binding cyto-
chrome a3 component of the mltochond-
nal ox~dase Further work ts necessary to
test tlus model
Some functional aspects of the
respiratory chain
Figure 3 shows a stmple scheme for the
functtonal organ~zatton of the compo-
nents of the resptratory chain and the
coupling sites for oxadatwe phosphoryla-
uon m E. cob Dehydrogomses act on
substrates on the inner surface of the
cytoplasmic membrane, and transfer the
eqmvalent of two hydrogens (2e- plus
2H +) to ublqmnone-8 which ss vatlun the
membrane.
The ubzqumol-8 generated by the
dehydrogenases ts presumably able to
diffuse laterally wtthm the membrane
and can also transfer reducing eqmva-
lents across the bdayer (see Ref 10)
Both of these features are built into the
model tn Fig 3 Either of the two
oxMases will oxidize ubiqmnol-8
Ubtqumone serves as a branch pomt m
the respiratory system, and can prowde
electrons to either oxadase The
ublqumol oxadal3on stte of the cyto-
chron,e d complex is located near the
penplasnuc (outer) surface of the mem-
brane and is assocaated primarily with
subumt I (cytc~hrome b55s) The oxida-
tion of ub|qumol releases two protons
(one H+/electron) The electrons are
then transferred through cytochrome
b595 to the other s~de of the membrane
where the 0 2 reducuon site is located,
associated with subumt I! (cytochrome
d) The electrogenlc transfer of electrons
ts respons|ble for generating the voltage
265
fer reactions to the generation of a trans-
H/CH2 3 membrane electrochemical proton gra-
dient, and the terminal oxldases which
perform thts funcuon can contain hemes
a, b or d (2) The resptratory chain of E
colt is mu:h less efficaent than the
mitochondnal chdln where at least three
protons are translocated per electron by
Complexes III and IV The E coh elec-
tron transfer system appears to be bruit
for adaptabdlty rather than efficiency
Condnsions
The apphcatlon of genetics, biochemi-
cal and biophysical techmques has
helped to answer many longstanding
questions concernmg the orgamzat~on of
OJ~'CXOH o~C"~OH the aerobic resp]ratory cham m E coh
The two terminal oxldases clearly are at
Fig 2 The structure proposed for the heine d
the heart of the E colt b~oenergeuc sys-
(chlorm) prosthenc group tn the cytochrome d com.
plex This ehlorm brads to 0 2 and Is presumably tem The proposed model ts not the only
located near the msute surface of the cytoplasmic one that fits the data, and must be ver-
membrane Thls group can be syntheslzed, m pnn- ified and filled m with cntscal detml con-
ctple, from protoheme IX One of the pyrrole rings cermng the way m which these enzymes
ts reduced, defmtug th~sas a chlorm, and two hydro. carry out two- and four- electron cherms-
xyl groups have been added try with hemes The question of mter-
mechate earner ~ystems serwng to fac~b-
difference across the membrane con- tate ubsqumone reducuon by some of the
conutant with enzyme turnover Finally,
the reduct|on of 02 to H20 utdtzes one
H + per electron on the cytoplasnuc
(tuner) side of the membrane The net
reaeUon Is the electrogemc transfer of
one H + across the membrane per elec-
tron In tins model, the enzyme does not
funcUon as an ton pump, as the cyto-
chrome c oxidases must, but, rather, as a
transmembrane electron transferase m
which the proton translocatlon results
from the chermcal reactmns that are
catalysed on the two sides of the mem-
brane. Note that m this model, ~t does
not matter whether the dehydrogenases
are located on the ms|de or outside of the
membrane Tius is consment with the
observations that purified dehydro-
genases can be coupled to ~ . respiratory
system when added to the outside of E
coh membrane vesicles, that ts, from the
wrong stde (see Ref 30)
Presumably, the stte of ubtqtanol oxi-
dation by the cytochrome o complex is
also near the outer surface of the mem-
brane If the analogy with the
mttochondnal oxldase ,s correct,
cyto-
chrome b562 would be expected to be
¢hrectly revolved m the oxzdatwe part of
the reacuon on the outer surface of the Ftg 3 A modelfortheaeroblcresplratorvsvstemm
membrane Copper could serve an elec- the cytoplasmic membrane of E cob A series of
tron transfer anti/or O2-bmdmg function, dehydrogenases reduce ublqumone-8, the product,
ubtqumol-8, diffuses wuhm the bdayer and is
and cytochrome o (b555)would be on the
oxl(hzed by either ternunal oxutase It t~ proposed
inner surface More data are reqmred to that the two oxtdase enzymes serve as coupling sites
test this model for oA~datl~e phosphoq'latlon and generate a proton
Two points are worth emphasizing motwe force by sepasatmg the two half reactwns on
(I) Nature has evolved several alternate opposue sutcs of the membrane, as dl~trated Q
mechamsms for couphng electron trans- and F denote ublqumone.8 and FAD, respecnvely
 266 TIBS 1 2 - July 1987

dehydrogenases ts another area winch
should be addressed, though there ts no
hard evadence for tins at the present
time
The last few years have clanfied the
overall orgamzattonal pattern of the
respiratory chain, and the system can
now be genettcally mampulated It was
100 years ago that German Imcro-
b~olog~st T Eschench found Bacterium
cob commune (renamed Eschertchta colt
later) The next few years, at the very
begmmng of the second century for E
cob, vail focus on testing models and
addressing questtons pertatmng both to
bacterial physiology and the enzymology
of tlus complex system
References
I Ke~hn,D (1933)Ergeb Enzymfors~h 2,239- 15 Green, G N Lorence,R M and Genres.
271
2 Poole,R K (1983)B~ochtm e~Bwphys Acta 16 Kranz,R G andGenms,R B (1984)./ Btol
726,205-243
3 Green,G N andGenms.R B (1983)./ Bac- 17 Ttmkovtch,R, Cork. M S. Genres,R B and
tenol 154,1269-1275
4 Au, D C-T, Lorence. R M and Genres,
R B (1985)./ Baacnol 161,123--127
5 Kranz,R G andGennts,R B (1985)J Bac-
tenol 161,709-713
6 IOta.K. Murakaml,H. Oya. H and Anraku.
Y (1985)Bmchem Int 10,319-326
7 Murakaml,H, IOta.K and Anraku.Y (1986)
J Bwl Chem 261.548--551
8 Mm'akam~, H , IOta, K and Anraku.Y (1984)
Mol Gee Genet 198,1--6
9 Wallace. B J and Young. J G (1977)
B~och~m B~ophys Acta 461,84-100
10 Hackenbrock,C R, Chazotte, B and Gupte,
S S (1986)l Bwenerg Bwmembranes 18,
331-368
I I IOta.K. Komsht,K and Anraku,Y (1984)J
Btol Chem 259,3375-3381
12 Mdler,M J andGenms,R B (1983)J Bwl
Chem 258, 9159-91o5
13 Green, G N. Kranz,J E and Genres, R B
(1984)Gene 32, 99-106
14 Lorence, R M Koland,J G and Genres,
R B (1986)B~oehem~stry25, 2314-2321
R B (1986)B~ochem~stry25, 2309--23,4
Chem 259,7998-8003
Johnson, P Y (1985)J Am Chem Soc 107.
6069-6075
18 Hata, A, Ionno, Y, Matsuura. K, Itoh, S.
Htyama,T, Komsln,K, Iota, K and Anraku.
Y (1985)Bmchtm Btoph.vs Acta 810,62-72
19 Koland,J G, Mdler,M J and Gennls,R B
(1984)Bmchermstry23 445-453
20 Mtller,M J andGenms.R B (1985)3 Biol
Chem 260. 14003--14008
21 Matsushtta. K, Patel, L, Gennls.R B and
Kaback, H R (1983)Proc Ned Acad Scl
USA 80, 4889-4893
22 Matsusluta.K Patel. L and Kaback H R
(1984) Bmchermstry23, 4703--4714
23 Iola, K, Komsht.K and Anraku,Y (1984)
J Btol Chem 259,3368-3374
24 Carter, K and Genres, R B (1985)J Btol
Chem 260. 10986-10990
25 Ktta,K, Kasahara.M and Anraku.Y (1982)
J Bml Chem 257,7933-7935
27 Poole,R K ,Salmon I andChance.B (1983)
J Gen Mtcroblol 129,1345-1355
28 Uno.T. Ntshlmura Y. Tsubol.M. gata. K
and Anraku, Y (1985) J Btol Chem 260,
6755--6760
29 Malmstrom.B G (1979)Biochlm Bmphys
Acta 549.281-303
30 Cronan, J E, Jr. Genres R B and Maloy,
S R (1987)m EschenchtdcOhandSalmonella
typhlmunumCellular and Molecular Bwlogy
(Netdhardt, F C, ed ), Vol 1, pp 31-35,
AmencanSocietyof Mtcrotnology

graduate student m the Eastern USA

Biochemistry of the neurotoxic synthesized for his own Intravenous
use MPPP (l-methyl-4-proplon-oxypip-
action of MPTP: endme), an analog of the narcottc petht-
dine (mependlne), later known as 'new
or how a faulty batch of'designer drug' led to heroin' MPTP is a variable by-product
parkinsonism in drug abusers of the synthesis, since MPPP readily
breaks down to MPTP at low pH or at
Thomas P. Singer, Anthony J. Trevor and Neal Castagnoli, Jr elevated temperature, while pethldlne
itselflschemlcaUy stable (Fig I). Inject-
m g a hurnedly prepared batch, he came
Less than four years ago, the sudden appearanoe o f parkmsontan symptoms m young drug down vath parkmsoman symptoms
abusers was traced to the presence of MPTP (l-methyl-4-phenyl-l,2,3,6-tetrahydropyndme) whlch responded to L-dopa treatment.
m carelessly synthesized batches of'new heroin', an dltctt pethulme analog The wodd-wule Researchers at the NaUonal Institute of
m~orestand interne research effort th~sd~covery generated has resulted m the M e n t i O n of Mental Health, puzzled by the appear-
the enzymes mvolved m ~zeconverston o f MP TI- ~ d~eneurotoxtcform It has also eluculated ance of a disease normally seen only m
~ e ouctal roles played by the dopanut~ reuptake system, by a newly dt~overed nutochon- aged patients, eventually traced the
dnal camer, and by nutochondnal N A D H dehydrogenase, the ulumate target, m the destruc- symptoms to his use of MPPP, contami-
tton of the mgrostnatal cells o f the brain nated w~th traces of MPTP The student
later died from a drug overdose, and on
The story of the idenuficatlon of MPTP Much of the pubhcity has focused on the autopsy extensive destruction of the sub-
as the toxtc agent responsible for the sud- formtdable social problems posed by the stantm mgra was found, as m id~opatinc
den onset of parkmsontan neurologtcal prohferatton of designer drugs - narco- parkmsomsm I The slgmhcance of these
symptoms m a group of heroin addtcts tics destgned and synthes~ed m order to findings was not appreciated at the time
who had rejected themselves with the circumvent the law, by shghtly altenng because expenments designed to test the
designer drug 'new heroin" has all the the structure of a controlled substance chj'omc neurotoxlc effects of mixtures of
elements of a good detective story It has without losing its narcotzc potency Little MPPP and MPTP m rats gave negatwe
been widely pubhclzed on radio, tele- has been written m the popular press, results The choice of species was unfor-
vision and in the press around the world however, of the remarkable success dur- tunate, since rats are now known to be
ing the past three years m tracing the remarkably resistant to the neurotoxlc
T P SmgertsmtheMolecularBiologyOlvcnon, Vet- senes of blochemtcal reactmns which effects of MPTP
eransAdrmnmmtwn MedicalCenter,San Franct~co, lead from the entry of MFTP into the The connection between MPTP anO
CA 94121, USA, and m ihe Depam,~entsof B~o. brain to the destructmn of dopammergtc parkmsoman symptoms was recognized
cher,usrryand Bmphy~csand Pharmacyat the Uru-
vmuy of Cahfomla. San Franctsco,N Castagnoh,Jr neurons in the substanua mgra of the early m 1982 when a number of relatwely
tcm the Depanmentof Phannacyand A Trevorts m mldbram, and, hence, to a plaustble ex- young patients were hoslmahzed m the
Ihe Departmentsof Pharmacology and Pharmacy, planauon of the neurological symptoms San Francisco Bay Area with sudden
Un,ve~iy of Cahfortua, San Franctsco,CA 94143, caused by MPTP onset of neurolog=cal symptoms closely
USA The story began m 1977 when a resembhng those seen m elderly subjects
~) lq87 ElsevierPubhc*huns Cdmbndce¢ 0376- 5~67t~7/~2.0U