HEMOGLOBIN, 1(8), 815-827 (1977)

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HAEMOGLOBIN A FRUSTRATED OXIDASE?
IMPLICATIONS FOR RED CELL METABOLISM

Rohin W. Carrel#, Christine C. Winterbourn*

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and John K. French*

fUni vers it y Department o f C1in i c a l Biochemistry ,
Addenbrooke's Hospital, Cambridge CR2 2QR, England
and *Pathology Department, Christchurch Hospital,
Christchurch, New Zealand

Abstract

The haem proteins can be considered, i n one aspect o f t h e i r

function, as machines f o r a c t i v a t i n g oxygen. I n the case o f
oxygen-carriers such as haemoglobin, the g l o b i n has evolved so
t h a t i t s conformation l i m i t s access t o the haem group, with
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r e s u l t a n t r e v e r s i b l e release o f oxygen. However, d i s t o r t i o n o f

the globin may allow e i t h e r the discharge o f oxygen as the
activated product superoxide or, more threateningly, allow d i r e c t
function o f the haemoglobin as an oxidative enzyme. Support f o r
t h i s i s shown by the reaction w i t h acetylphenylhydrazine where
haemoglobin functions as both an oxidase and oxygenase. An
i m p l i c a t i o n o f oxidase a c t i v i t y i s the p o t e n t i a l t o i n i t i a t e
f r e e r a d i c a l formation p a r t i c u l a r l y w i t h unsaturated l i p i d s .
Observations o f the acetylphenylhydrazine reaction emphasize the
r o l e o f glutatliione as a free r a d i c a l scavenger.

Introduction

I t i s tempting t o t h i n k o f haemoglobin, w i t h i t s elegant

co-operative effects, as being c a r e f u l l y designed as an oxygen

carrier. Yet it, l i k e other protejns, has evolved i n a series

-~ ~-

Received: May 27, 1977; Accepted: October 4, 1977

815
Copyright @ 1978 hy Marcel Dekker. Inc. All Righlr Reserved. Neither ihir work nut any par1
muy be reproduced or lranrmillcd in any form or by any means. electronic or mechanical. including
pholocopying. microfilming. and recordins, or hy any information blorasr and rrlricval sytlrm.
wilhoul permission in writing from the publisher.
 816 CARRELL, WINTERBOURN, AND FRENClI

o f makeshift 'mutations from more p r i m i t i v e proteins w i t h q u i t e

different.functions. As with a l l makeshift changes there i s the

r i s k t h a t s l i g h t disruption may cause a reversion t o the e a r l i e r

function. Clearly, i t would be an advantage t o understand the

p r i m i t i v e 'precursor' functions o f haemoglobin as t h i s could

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give a key t o the processes t h a t accompany haemoglobin denatur-

a t i o n as I n the thalassaemias, the unstable haemglobins, and

the oxidative haemolytic anaemias i n general.

Oxyhaenoglobin - A F e r r i c Superoxide
The ultimate precursor o f haemoglobin must belthe haem

group i t s e l f . This has an obvious redox function, i.e. i t can

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p o t e n t i a l l y a c t as both a donor and acceptor o f electrons. The

conplex o f loosely-bound H electrons o f the protoporphyrin allow

'a metal c b o r d i n a t e t o undergo changes i n charge as i n the

ferrous t o f e r r i c transitions o f the haem iron. Weias (1) pointed

out i n 1964 t h a t t h i s a b i l i t y t o undergo charge transitions would

a l l o w oxygen t o reversibly complex w i t h the ferrous i r o n as the

f e r r i c superoxide, i n which the i r o n i s i n (or near) the f e r r i c

stage and an electron i s transferred t o the oxygen t o give a

superoxide ion.

(Hb)Fe2+ + O2 * (Hb)Fe3+ 0; I

A proviso being t h a t the haem group i s i n an apolar environment.

as i s provided by the globin molecule.
 HAEMOGLOBIN - A FRUSTRATED OXIDASE? 817

Although the concept o f oxyliaemglobin as a f e r r i c super-

oxide i s now generally accepted (2,3,4) i t i n i t i a l l y received

l i t t l e support from the structural chemists. However, the

proposal had an inmediate a t t r a c t i o n t o those a t the time who

were studying the changes observed i n the unstable haemoglobin

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diseases (5,6). This was because Weiss's proposals implied t h a t

a disruption o f the globin haem pocket would r e s u l t i n displace-

ment o f the superoxide i o n giving both methaemglobin fornation

and an accompanying oxidative stress.

(Hb)Fe3+ 0; * (Hb)Fe3+ + 0; I1

This provided a s a t i s f y i n g explanation f o r the chanqes i n red c e l l

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metabolism seen i n the unstable haemoglobin diseases which are

associated w i t h increased methamglobin formation bnd glutathione

(GSH) consumption (5). The oxidative stress follows from the

release o f superoxide; t h i s i s the f i r s t step i n the production o f

a group o f oxygen derivatives known as activated oxygen which

includes hydrogen peroxide, the hydroxyl free radical and s i n g l e t

oxygen.

Activated Oxygen
Oxygen i t s e l f i s r e l a t i v e l y unreactive and i t i s not u n t i l i t

i s raised t o an activated form t h a t oxidation o f biological

molecules i s l i k e l y t o occur. The unsaturated membrane l i p i d s are

p a r t i c u l a r l y a t r i s k and hence the release o f superoxide and

subsequent formation o f f u r t h e r activated oxygen i s a potential

 818 .
CARRELL WINTERBOURN, AND FRENCll

contributor to, red c e l l haemolysis i n the haemoglobinopathies

associated w i t h premature denaturation (7). This i s especially

true o f the thalassamias where the isolated chains r e a d i l y

denature and where the organelles o f the i m t u r e c e l l s provide

p a r t i c u l a r l y susceptible targets (8).

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D i r e c t evidence o f superoxide release i n confirmation o f

Weiss's proposal has been provided by Misra and Fridovich (9)
and demonstrated t o be increased i n unstable haemoglobins and

thalassaemias by others (10,ll). However, the l i m i t e d studies

t h a t have so f a r been c a r r i e d out raise some doubts tas t o whether

superoxide formation i s an appreciable cause o f red c e l l damage.

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Adequate defences e x i s t i n the form o f superoxide dismutase.

catalase and the glutathione peroxidase system. Furthermore, any

superoxide escaping these defences should predominantly react w i t h

haemglobin which i t does i n a rather benign and reversible manner

(11). Other more threatening p o s s i b i l i t i e s e x i s t such as the

reactfon of superoxide and hydrogen peroxide i n the presence o f

free i r o n t o give the highly reactive hydroxyl radical. These are

p o s s i b i l i t i e s , bud the indications are t h a t we may have t o l o o k

beyond activated oxygen t o the possi b i lity t h a t haemoglobin i s

reverting. i n these circumstances, t o i t s p r i m i t i v e r o l e as a

d i r e c t oxidase o r oxygenase.

Haemoglobin as an Oxidase

A f i r s t requirement i n a p r i m i t i v e aerobic organism would be

the a b i l i t y t o carry out the controlled oxidation o f organic
 HAEMOGLOBIN - A FRUSTRATED OXIDASE? 819

substrates. This can e i t h e r occur by hydrogen abstraction, as i n

the oxidases, o r by d i r e c t addition o f oxygen as i n the oxygenases,

hydroxylases and Cyt P450 etc. The s i t u a t i o n i s i l l u s t r a t e d i n

Figure 1 with reference t o the oxygenases. The first function o f

the haem enzyme i s that o f oxygen activation as i n A, followed by

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Figure 1

Both the Oxygt3Iases and haemglobin share a comuon primary

reaction with oxygen, a c t i v a t i o n as i n A. I n the oxygenares the
activated oxygen Is used f o r s u b s t r a t e oxidation as i n B but
r e v e r s i b l e r e l e a s e of oxygen can occur as i n C. I n haemglobin
the physiological function is t h a t of cycle AC but d i s t o r t i o n
of the globin may allow reversion t o an oxidative r o l e as i n AB.
 820 CARRELL, WINTERBOURN, AND FRENCH

substrate oxidation, i n t h i s case scission o f a double bond, as i n

0. A good example o f such an enzyme would be the indoleamine-2,3-

dioxygenase described by 0. Hayaishi and colleagues (personal

conmunication). This enzyme gives a clue t o the evolutionary

pathway o f haelnoglobin as i t can not only catalyse the scission o f

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indoleamine but i t can also function, as i n C o f Figure 1, as a

reversible oxygen carrier.

This type o f enzyme would c l e a r l y provide the basis f o r

evolutionary development o f the primary oxygen carriers required

by larger organisms. The f i r s t requirement f o r the oxygen

c a r r i e r s being a conformation t h a t l i m i t s access t o the haem

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pocket by substrates other than oxygen and that provides a low

d i e l e c t r i c environment around haern. Alterations o f the s t e r i c

structure o f the haern pocket could thus change the function o f the

molecule from an enzyme t o t h a t o f an oxygen-carrier and v i c e -

versa.

I t i s t h i s reverse p o s s i b i l i t y , the a l t e r a t i o n from oxygen

c a r r i e r t o oxidase (oxygenase), t h a t p a r t i c u l a r l y interests us.

This could occur i n two situations. The f i r s t i s t h a t of

denatured h a m g l o b i n , as i n isolated chains, where major conform-
ational changes a t the h a m pocket could allow the haemoglobin t o

function as a d i r e c t oxidase o f large molecules such as membrane

lipids. This i s s t i l l hypothetical, although i t would provide a

neat explanation f o r some o f the features of the oxidative

 HAEMOGLOBIN -A FRUSTRATED OXIDASE? 82 1

anaemias n o t explicable i n terms o f activated oxygen. However,

firm evidence i s now appearing f o r the second s i t u a t i o n i n which

haemoglobin can function as an oxidase. This i s with small

molecules such as drugs t h a t can gain d i r e c t access t o the haem

pocket o f n a t i v e haemoglobin. The best studied example i s the

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d i r e c t oxidation o f acetylphenylhydrazine by oxyhaemoglobin.

Reaction with Acetylphenylhydrazine

Acetylphenylhydrazine, APH. i s t y p i c a l o f a v a r i e t y o f redox

drugs that react w i t h haemglobin causing denaturation, p r e c i p i t -

a t i o n as Heinz bodies, and accelerated red c e l l destruction.

With each drug the o v e r a l l reaction consists o f a large number o f

interdependent steps. b u t there i s increasing evidence (12) t h a t

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the f i r s t step i s reaction 1 1 1

Here the generalised drug has been represented as RH2 b u t s p e c i f i c

reference w i l l be made t o APH (Formula $ below).

C6 H5-NHNHCOCH3 C6H5-N'NHCOCH3 C6H5-N=NCOCH3

-a -b -
C

APH - radical - diazine

Reaction I I I can be considered as the haemglobin-catalysed

autoxidation of the drug, w i t h the oxygen being activated i n super

-0xoferri-haemoglobin which functions as an oxidase. The oxygen

i s reduced t o H202 by t h e t r a n s f e r o f 2 electrons. one from the

haern iron, producing methaentoglobin, and one from the APH forming
 822 CARRELL. WINTERBOURN, AND FRENCH

the drug free radical (b above) o r i n general RH'. Thus, the

f i r s t (oxidase) step o f the reaction produces methaemoglobin and

H202, i n i t i a l l y as a complex. However, i n addition, a highly

reactive free radical i s formed which appears t o play a crucial

p a r t i n determining the course o f the reaction. One end product

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of the reaction i s choleglobin, formed by attack o f the complexed

H202 on the porphyrin r i n g - an example o f oxygenase a c t i v i t y by

haemglobin. The other product i s haemichromes formed by the

binding of the diazine (c above) t o methaemoglobin as proposed by

Itano and colleagues (13,14). The detailed supporting evidence

f o r t h i s a l m g w i t h the factors a f f e c t i n g each step are given by

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French e t a1 (12). The sequence of changes t o the haemglobin i s

sunmnarised i n equation I V .

-I haemichrome %

Hb02 + APH + metHb ~ ~ precipitate IV

choleglobin

The f i r s t step being an example o f oxidase a c t i v i t y , the second

( t o choleglobin) o f t h a t o f an oxygenase.
Role o f Glutathione and Ascorbate
A c l i n i c a l l y s i g n i f i c a n t f i n d i n g i s that the addition o f

reduced glutathione (GSH) slows down the overall r a t e o f the

reaction by decreasing the r a t e of oxyhaemoglobin oxidation. This

protection by GSH occurs i n the absence o f GSH peroxidase, i n the

presence o f high catalase concentrations, and a t GSH: haemglobin
r a t i o s s i m i l a r t o those i n the red c e l l . A s i m i l a r slowdown i n
 1IAT:MOCI~OIJIN -A FRUSTRATED OXLMSE? 023

the r a t e o f oxidation i s also observed i n the presence o f

ascorbate. Both ascorbate and GSH are reducing agents, hut they

are also e f f i c i e n t f r e e r a d i c a l scavengers (15). and a l l the

evidence indicates t h a t each i s acting as a scavenger i n t h i s

system as i n reaction V.
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APH radical t GSH + APH t GS' + GSSG V

The o v e r a l l reaction i s i n h i b i t e d because GSH competes f o r the APH

r a d i c a l and prevents i t reacting w i t h more oxyhaemoglobin as i n

reaction V I .

APH radical t Hb02 -L Hb3' + diazine t H202 VI

This scavenging of the APH r a d i c a l also decreases the amount o f

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diazine formed, so there i s less available f o r the formation o f

unstable haernichromes. GSIl and ascorbate also i n h i b i t other

reactions a t t r i b u t a b l e t o the APH radical, including the reaction

w i t h O2 t o give superoxide (16,17) and reaction with u n i d e n t i f i e d

s i t e s on the globin, producing even less stable haemichromes (12).

The demonstration t h a t GSH, a c t i n g as a f r e e radical

scavenger, can i n h i b i t the breakdown o f oxyhaemoglobin by APH

must be considered i n r e l a t i o n t o the normal r o l e o f GSH i n the

red c e l l . Although some years ago Kosower and Kosower (18) drew
a t t e n t i o n t o the f r e e r a d i c a l scavenuing c a p a b i l i t y of GSH, t h i s

has tended t o be overshadowed by i t s r o l e i n the GSlf peroxidase-

catalysed removal of H202. The l i n k between drug s e n s i t i v i t y and

G6PD deficiency i s generally considered as an i n a b i l i t y t o

 824 CARRELL, WINTERBOURN, AND FRENCi

remove the excess H202 t h a t i s generated. However, the drugs

t h a t are haemolytic i n G6PD deficiency produce f r e e radicals on

reaction w i t h haemoglobin. These r a d i c a l s accelerate haemichrome

and choleglobin formation, and can form damaging hyperoxides

with membrane phospholipids (15). Thus, the prime r o l e o f GSH

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may be as a f r e e r a d i c a l scavenger and t h a t o f glutathione

peroxidase as a l i p i d hydroperoxidase (19).

Concl us ions

The concept o f haemoglobin as an oxidase/oxygenase i s

implied i n the e a r l i e r w r i t i n g s o f R. Lemberg and J . J. Weiss and

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supported more recently by others (20.21.22). O f particular

i n t e r e s t , t o us, i s the p o s s i b i l i t y t h a t t h i s a c t i v i t y might be

potentiated by perturbation o r denaturation o f the haemoglobin

molecule.

The mechanisms involved i n the haemolysis associated w i t h

the i n c l u s i o n body anaemias, such as thalassaemia, are s t i l l ill-

understood. There i s good evidence t h a t o x i d a t i v e damage i s

occurring i n conjunction w i t h haemoglobin denaturation. This

could be due t o the release o f a c t i v a t e d oxygen b u t r e d c e l l

p r o t e c t i v e mechanisms should s u f f i c e t o prevent major damage.

However, a s i t u a t i o n t h a t could not be r e a d i l y guarded against i s

the d i r e c t a c t i v i t y o f denatured haemoglobin as an oxidase on

substrates such as membrane phospholipids. The close association

 HAEMOGLOBIN -A FRUSTRATED OXLDASE? 825

o f denatured haemoglobin w i t h c e l l membranes, which i s seen i n

the thalassaemias and unstable haemoglobin diseases, reinforces

the p o s s i b i l i t y t h a t d i r e c t oxidation may be a contributory cause

o f c e l l lysis.

Acknowledgments
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We g r a t e f u l l y acknowledge the encouragement given t o us by

Professor H. Lehmann, F.R.S. t o enter and continue i n the f i e l d

o f research o u t l i n e d i n t h i s review. The work was supported i n

p a r t by the Medical Research Council o f New Zealand.

References
For personal use only.

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2) Maxwell, J. C. and Caughey, W. S., Biochem. Biophys. Res.

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K. R., Proc. tiatl. Acad. Sci. U.S.A. -

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6) Lehmann, H., i n Fourth Symposium o f Advanced Medicine,

edited by 0. Wrong, p. 219, Pitman Medical London, 1968.

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 826 CARRELL, WINTERBOURN. AND FRENQI

Rachmilewitz, E. A., Lubin, B. H. and Shohet, S. B., Blood,

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a c t i v a t e d oxygen i n drug induced haemoglobin breakdown.

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B. Med. Sc. Thesis, U n i v e r s i t y o f Otago, New Zealand).

13) Itano, H. A., Proc. Natl. Acad. Sci., U.S.A. , 2: 485, 1970.

14) Itano, H. A., Hirota, K. and Hosokawa, K., Nature, 256: 665,

1975.

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published Pion Ltd., London, 1972.

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S., Nature 224: 117, 1969.
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J., 139: 289, 1974. '

 HAEMOGLOBIN - h FRUSTMTED OXIDASE? 827

20) Yamazaki, I . i n Hayaishi, ed., Molecular Mechanisms o f

Oxygen Activation, Academic Press.

21) Mieyal, J. J . , Ackerman, R. S. and Rlumer, J . L . , J. Biol.

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159: 23, 1976.
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