A . HYDROGEN PEROXIDE
There is evidence t h a t hydrogen peroxide m a y serve as t h e ultimate oxi
d a n t of t h e cytochrome chain in bakers y e a s t ,1 2 0 , 1 2 1 Acetobacter peroxi-dans,m~m a n d Pseudomonas fluorescens.m T h e first evidence on this point came when a peroxidase for reduced cytochrome c was isolated a n d highly purified from yeast.1 2 0 T h e enzyme, which contains a n iron protoporphyrin prosthetic group, was reported t o be rather specific for cytochrome c as substrate. Chance,1 2 6 however, reports t h a t typical peroxidase substrates, such as p-phenylenediamine, can be oxidized b y t h e enzyme and, further, t h a t t h e classic peroxidases of plant a n d animal origin also have high
ac-340 Μ. I. DOLIN
tivity with reduced cytochrome c as substrate. I n addition, he has shown t h a t in intact respiring yeast, exogenous H2O2 is utilized even more rapidly t h a n oxygen for the oxidation of reduced endogenous cytochrome c.1 2 1
Acetobacter peroxidans122'124 can use H2O2 as the oxidant for several sub
strates, including H2 . A cytochrome peroxidase mechanism m a y be present in these cells.1 2 3 A cytochrome c peroxidase has been isolated from P. fluor
esceins?2* the partially purified enzyme contains a cytochrome c pigment, however, the prosthetic group of the peroxidase itself is unidentified. Since P. fluoresceins appears to contain a flavoprotein D P N H oxidase, it has been suggested t h a t the following sequence of reactions describes t h e respir
atory system of the organism.
D P N H + H+ + 02 H202 + DPN+ (DPNH oxidase) (11a) D P N H + 2 cyt. c (Fe+++) —
DPN+ + 2 cyt. c ( F e+ +) + H+ (cyt. c reductase) (lib) 2 cyt. c (Fe++) + 2 H+ + H202 ->
2 cyt. c (Fe+++) + 2 H2Q (cyt. c peroxidase) (11c) Sum: 2 D P N H + 2 H+ + 02 -> 2 H20 + 2DPN+
This scheme, which m a y be taken as a model for peroxidase-mediated respiration, illustrates one difficulty involved in trying to assess t h e physio
logical significance of iron-porphyrin peroxidases, namely, in order for t h e enzyme to function, a source of peroxide must be present. As shown in the scheme, the source is generally attributed to t h e autoxidation of flavopro
teins. T h e question which has not yet been satisfactorily answered for a n y of t h e cytochrome-containing organisms is the extent t o which such autoxi
dation reactions m a y fulfill the requirement of a peroxide-generating sys
tem. I n yeast1 2 1 it is believed t h a t flavoproteins are not autoxidized rapidly enough to play the role required b y equations ( l l a ) - ( l l c ) . E v e n if a bypass to oxygen via flavoprotein does exist in a given system, the reaction need not yield p e r o x i d e .4 7 5 0 6 7"6 9 The physiological role of these interesting per
oxidases remains to be clarified. T h e role of H2O2 in the respiratory systems of cytochrome-free bacteria will be considered in Chapter 9.
B . NITRATE, NITRITE, SULFATE, ETC.
Various lines of evidence have implicated: (a) cytochromes of the b-type in the reduction of nitrate by P. denitrifieans,** M. denitrificans,® a n d E.
coli,121'128 and (b) cytochromes of t h e c-type in the reduction of nitrate b y Μ. denitrificans12* and A. fischerim and in t h e reduction of nitrate and ni
trite b y P . aeruginosa™ A c-type cytochrome is implicated in sulfate, thiosulfate, and sulfite reduction in the anaerobe Desulfovibrio desulfuri-cans.m*132a A general formulation of such reactions is shown in Fig. 6.
6. MICROBIAL ELECTRON TRANSPORT MECHANISMS 341
FIG. 6. Reduction of inorganic nitrogen and sulfur compounds. The true sub
strate for the cell-free sulfate reductase of Desulfovibrio desulfuricans is adenosine
s' -phosphosulf a t e .1 3 2 0
(Evidence for pyridine nucleotide a n d flavin mediation has been reported for t h e nitrate r e d u c t a s e s ,1 2 7 , 1 2 9 and for nitrite and nitrous oxide reduction,1 3 2 1* schemes such as t h a t shown in Fig. 6 account for the "nitrate respiration"
of various organisms (E. coli, Pseudomonas, S. aureus) t h a t are able t o grow anaerobically with nitrate as an electron acceptor. According t o this view, flavoproteins t h a t utilize nitrate as t h e immediate oxidant4 8 m a y be spe
cifically involved in t h e nitrate assimilation reactions, b u t not in "nitrate respiration."
There is some question whether specific reductases are always needed for t h e reoxidation of reduced cytochrome b y inorganic nitrogen com
pounds. Cytochrome c3 of Desulfovibrio, for instance, is reoxidized nonen-zymically b y N H2O H .1 3 3 I t is possible t h a t t h e reoxidation of Μ. denitri-ficans cytochrome b by nitrate is a spontaneous reaction.6 3
C. ARTIFICIAL ELECTRON ACCEPTORS
Electron flow m a y be diverted to artificial acceptors such as oxidation-reduction d y e s ,1 3 4 , 1 3 5 quinones,8 1 and iron compounds such as ferricyanide.1 3 6 These acceptors are useful when t h e natural physiological acceptor is un
known or not easily available. Dyes which undergo color change on reduc
tion (bleaching of methylene blue, or 2,6-dichlorophenol-indophenol) are especially useful. T h e methylene blue technique,1 3 7 for instance, was of great practical importance in early studies of bacterial enzymes. Enzymes studied (anaerobically) with such acceptors were operationally defined as dehydrogenases since the reactions presumably involved transfer of (2H) from substrate t o artificial acceptor, a n d did not require t h e participation of oxygen. A dehydrogenase, defined in this way, m a y consist of a complex
342 Μ. I. DOLIN
of enzymes and intermediate carriers. Since artificial acceptors are not specific for a given locus, the composition of a particular "dehydrogenase"
system cannot be deduced solely from the observation t h a t one or another acceptor m a y function as oxidant. For instance, various oxidation-reduction dyes, such as methylene blue or 2,6-dichlorophenol-indophenol,4 6'4 7 or quinones8 1 m a y act as oxidants for flavoprotein. I n the succinic oxidase system, however, methylene blue does not function a t the primary dehy
drogenase (flavoprotein) site, b u t farther along in the c h a i n .1 3 8*1 3 9 Ferricya-nide m a y oxidize reduced flavoproteins19,47 or spontaneously oxidize reduced cytochrome.1 4 0 2,6-Dichlorophenol-indophenol a t p H 5.5 rapidly oxidizes D P N H in a spontaneous reaction;4 7" various quinones can oxidize D P N H spontaneously.1 4 1 Other nonenzymic oxidations of D P N H have been demonstrated and t h e rates tabulated.1 4 2 Although artificial electron accep
tors are not diagnostic reagents, they are specific to the extent t h a t they will not act as stoichiometric acceptors unless their oxidation-reduction po
tential is higher t h a n t h a t of t h e electron donor system (see Section V I I , A).
I t should be mentioned here t h a t when an autoxidizable acceptor is used, A H2 + oxidant —• oxidant · Η2 -f A (dehydrogenation) (12a) oxidant · Η2 + 02 —• oxidant + H202 (autoxidation) (12b) Sum: AH2 + 02 -> H202 + A
(methylene blue, some quinones, free flavins) the "dehydrogenase" can be coupled to oxygen, and an artificial "oxidase" t h u s created.1 3 4 Peroxide is the product of such reactions.1 3 6