Cytochrome o (cyoABCDE) and d (cydAB) oxidase gene expression in Escherichia coli is regulated by oxygen, pH, and the fnr gene product.

The aerobic respiratory chain of Escherichia coli contains two terminal oxidases that catalyze the oxidation of ubiquinol-8 and the reduction of oxygen to water. They are the cytochrome o oxidase complex encoded by cyoABCDE and the cytochrome d oxidase complex encoded by cydAB. To determine how these genes are regulated in response to a variety of environmental stimuli, including oxygen, we examined their expression by using lacZ protein fusions in wild-type and fnr mutant strains of E. coli. Anaerobic growth resulted in a 140-fold repression of cyoA'-'lacZ expression relative to aerobic growth and a 3-fold increase in cydA'-'lacZ expression. Anaerobic repression of both fusions was mediated in part by the fnr gene product, as evidenced by a 30-fold derepression of cyoA'-'lacZ expression and a 4-fold derepression of cydA'-'lacZ expression in an fnr deletion strain. Supplying wild-type fnr in trans restored wild-type repression for both fusions. Fnr thus functions as an anaerobic repressor of both cyoABCDE and cydAB expression. Reduced-minus-oxidized difference spectrum analyses of cell membranes confirmed the effect of the fnr gene product on the production of cytochrome d oxidase in the cell. Based on the pattern of anaerobic cydAB expression observed, we propose the existence of a second, as yet unidentified, regulatory element that must function either to activate cydAB expression as oxygen becomes limiting or to repress cydAB expression aerobically. Whereas cytochrome o oxidase encoded by cyoABCDE appears to be produced only under oxygen-rich growth conditions, in keeping with its biochemical properties, cytochrome d oxidase is expressed moderately aerobically and is elevated yet further when oxygen becomes limiting so that the organism can cope better under oxygen starvation conditions. We also examined cyoABCDE and cydAB expression in response to growth on alternative carbon compounds and to changes in the culture medium pH and osmolarity.     

The purification and characterization of the cytochrome d terminal oxidase complex of the Escherichia coli aerobic respiratory chain

The aerobic respiratory chain of Escherichia coli is branched. In aerobically grown cells harvested in midexponential phase, a respiratory chain containing only b-type cytochromes is predominant. This chain contains a terminal oxidase which is a b-type cytochrome, referred to as cytochrome o. However, when the bacteria are grown under conditions of oxygen limitation, additional components of the respiratory chain are induced, as evidenced by the appearance of new spectroscopic species. These include a new b-type cytochrome, cytochrome b558, as well as cytochrome a1 and cytochrome d. In this paper, a purification protocol and the initial characterization of the terminal oxidase complex containing cytochrome d are reported. Solubilization of the membrane is effected by Zwittergent 3-12, and purification is accomplished by chromatography with DEAE-Sepharose CL-6B and hydroxyapatite. The complex contains cytochrome b558, a1, and d. Analysis by sodium dodecyl sulfate-polyacrylamide gels indicates that the complex contains only two types of polypeptides with the molecular weights estimated to be 57,000 and 43,000. The purified complex has oxidase activity in the presence of detergents, utilizing substrates including ubinquinol-1, N,N,N',N'-tetramethyl-p-phenylenediamine, and 2,3,5,6-tetramethyl-p-phenylenediamine. The cytochrome d complex contains protoheme IX and iron, but does not contain nonheme iron or copper. Approximately half of the cytochromes which are thought to participate in E. coli aerobic respiration are accounted for by this single complex. These results suggest that the E. coli aerobic respiratory chain is organized around a relatively small number of cytochrome-containing complexes.     

Nitrate,fumarate, and oxygen as electron acceptors for a late step in microbial heme synthesis

Nitrate can serve as anaerobic electron acceptor for the oxidation of protoporphyrinogen to protoporphyrin in cell-free extracts of Escherichia coli grown anaerobically in the presence of nitrate. Two kinds of experiments indicated this: anaerobic protoporphyrin formation from protoporphyrinogen, followed spectrophotometrically, was markedly stimulated by addition of nitrate; and anaerobic protoheme formation from protoporphyrinogen, determined by extraction procedures, was markedly stimulated by addition of nitrate. In contrast, anaerobic protoheme formation from protoporphyrin was not dependent upon addition of nitrate. This was the first demonstration of the ability of nitrate to serve as electron acceptor for this late step of heme synthesis. Previous studies with mammalian and yeast mitochondria had indicated an obligatory requirement for molecular oxygen at this step.In confirmation of our previous preliminary report, fumarate was also shown to be an electron acceptor for anaerobic protoporphyrinogen oxidation in extracts of E. coli grown anaerobically on fumarate. For the first time, anaerobic protoheme formation from protoporphyrinogen, but not from protoporphyrin, was shown to be dependent upon the addition of fumarate.The importance of these findings is 2-fold. First, they establish that enzymatic protoporphyrinogen oxidation can occur in the absence of molecular oxygen, in contrast to previous observations using mammalian and yeast mitochondria. Secondly, these findings help explain the ability of some facultative and anaerobic bacteria to form very large amounts of heme compounds, such as cytochrome pigments, when grown anaerobically in the presence of nitrate or fumarate. In fact, denitrifying bacteria are known to form more cytochromes when grown anaerobically than during aerobic growth.An unexpected finding was that extracts of another bacterium, Staphylococcus epidermidis, exhibited very little ability to oxidize protoporphyrinogen to protoporphyrin as compared to E. coli extracts. This finding suggests some fundamental differences in these two organisms in this key step in heme synthesis. It is known that these two facultative organisms also differ in that E. coli synthesizes cytochrome during both aerobic and anaerobic growth, while Staphylococcus only synthesizes cytochromes when grown aerobically.     

Membrane-bound respiratory of Spirillum itersonii.

The membrane-bound respiratory system of the gram-negative bacterium Spirillum itersonii was investigated. It contains cytochromes b (558), c (550), and o (558) and beta-dihydro-nicotinamide adenine dinucleotide (NADH) and succinate oxidase activities under all growth conditions. It is also capable of producing D-lactate and alpha-glycerophosphate dehydrogenases when grown with lactate or glycerol as sole carbon source. Membrane-bound malate dehydrogenase was not detectable under any conditions, although there is high activity of soluble nicotinamide adenine dinucleotide: malate dehydrogenase. When grown with oxygen as the sole terminal electron acceptor, approximately 60% of the total b-type cytochrome is present as cytochrome o, whereas only 40% is present as cytochrome o in cells grown with nitrate in the presence of oxygen. Both NADH and succinate oxidase are inhibited by azide, cyanide, antimycin A, and 2-n-heptyl-4-hydroxyquinoline-N-oxidase at low concentrations. The ability of these inhibitors to completely inhibit oxidase activity at low concentrations and their effects upon the aerobic steady-state reduction levels of b- and c-type cytochromes as well as the aerobic steady-state reduction levels obtained with NADH, succinate, and ascorbate-dichlorophenolindophenol suggest that presence of an unbranched respiratory chain in S. itersonii with the order ubiquinone leads to b leads to c leads to c leads to oxygen.     

Recent studies of the cytochrome o terminal oxidase complex of Escherichia coli

The cytochrome o complex is the predominant terminal oxidase in the aerobic respiratory chain of Escherichia coli when the bacteria are grown under conditions of high aeration. The oxidase is a ubiquinol oxidase and reduces molecular oxygen to water. Electron transport through the enzyme is coupled to the generation of a protonmotive force. The purified cytochrome o complex contains four or five subunits, two protoheme IX (heme b) prosthetic groups, plus at least one Cu. The subunits are all encoded by the cyo operon. Sequence comparisons show that the cytochrome o complex is closely related to the aa3-type cytochrome c oxidase family. Gene fusions have been used to define the topology of each of the gene products. Subunits I, II, III and IV are proposed to have 15, 2, 5 and 3 transmembrane spans, respectively. The fifth gene product (cyoE) encodes a protein with 7 membrane spanning segments, and this may also be a subunit of this enzyme. Fourier transform infrared spectroscopy has been used to monitor CO bound in the active site where oxygen is reduced. These data provide definitive proof that the cytochrome o complex has a heme-copper binuclear center, similar to that present in the aa3-type cytochrome c oxidases. Site-directed mutagenesis is being utilized to define which amino acids are ligands to the heme iron and copper prosthetic groups.     

Identification of Reo1, a Gene Involved in Negative Regulation of Cox5b and Anb1 in Aerobically Grown Saccharomyces Cerevisiae

In Saccharomyces cerevisiae, the COX5a and COX5b genes constitute a small gene family that encodes two forms of cytochrome c oxidase subunit V, Va and Vb, either of which can provide a function essential for cytochrome c oxidase activity and respiration. In aerobically grown wild-type yeast cells, Va is the predominant form of subunit V. The COX5b gene alone does not produce enough Vb to support a respiration rate sufficient to allow growth on nonfermentable carbon sources. By selecting for mutations that increase the respiratory capacity of a strain deleted for COX5a, we have identified a gene that is involved in negative regulation of COX5b expression under aerobic growth conditions. Each of four independently isolated reo1 mutations are shown to be recessive, unlinked to COX5b, but dependent on COX5b for phenotypic expression. The mutations define a single complementation and linkage group: designated as REO1 for regulator of expression of oxidase. reo1 mutations increase expression of COX5b in aerobically grown cells, but not in anaerobically grown cells, where expression is already elevated. These mutations have no effect on COX5a, the other member of this small gene family which is positively regulated by heme and oxygen. The REO1 gene does play a role in repression of ANB1, a gene that is normally repressed under aerobic but not anaerobic conditions. Neither rox1 or rox3 mutations, which have previously been shown to increase ANB1 expression, are in the same complementation group as reo1 mutations.     

Electron Transport System of the Protoheme-requiring Anaerobe Bacteroides melaninogenicus

Protoheme is essential for the growth of some strains of Bacteroides melaninogenicus. At low concentrations in the growth medium, protoheme determines the doubling time, total cell yield, and amount of cytochrome per bacterium. At high protoheme concentrations, the doubling time, total cell yield, and amount of enzymatically reducible cytochrome appear to remain nearly constant, and protoheme is accumulated by the cell. The accumulated protoheme can support the growth of the bacterium for at least eight generations in a protoheme-free medium. When growth and cytochrome content are proportional during growth at low protoheme concentrations, the bacteria incorporate 10 to 20% of the total available protoheme into a membrane-bound respiratory system. This respiratory system includes cytochrome c, a carbon monoxide-binding pigment, and possibly flavoproteins. The pigments can be reversibly reduced by reduced nicotinamide adenine dinucleotide or endogenous metabolism and can be oxidized anaerobically by fumarate or by shaking in air. Electron transport is inhibited by 2-n-nonyl-4-hydroxy-quinoline-N-oxide.     

Coulometric and spectroscopic analysis of the purified cytochrome d complex of Escherichia coli: evidence for the identification of "cytochrome a1" as cytochrome b595

Coulometric and spectroscopic analyses were performed on the three cytochrome components (cytochrome d, cytochrome b558, and the cytochrome previously described as cytochrome a1) of the purified cytochrome d complex, a terminal oxidase of the Escherichia coli aerobic respiratory chain. On the basis of heme extraction, spectroscopic, and coulometric data, the "cytochrome a1" component was identified as a b-type cytochrome: cytochrome b595. The pyridine hemochromogen technique revealed the presence of two molecules of protoheme IX per cytochrome d complex. This quantity of protoheme IX fully accounted for the sum of the cytochrome b558 and cytochrome b595 components as determined coulometrically. The renaming of cytochrome a1 as cytochrome b595 was further indicated by the lack of any heme a in the complex and by its resolved reduced-minus-oxidized spectrum. The latter was found to be similar to that of cytochrome c peroxidase, which contains protoheme IX. Coulometric titrations and carbon monoxide binding titrations revealed that there are two molecules of cytochrome d per complex. A convenient measurement of the amount of cytochrome b558 was found to be the beta-band at 531 nm since cytochrome b558 was observed to be the only component of the cytochrome d complex with a peak at this wavelength. By use of this method and the extinction coefficient for the purified cytochrome b558, it was estimated that there is one molecule of cytochrome b595 and one of cytochrome b558 per cytochrome complex.     

Intracellular expression of Vitreoscilla hemoglobin modifies microaerobic Escherichia coli metabolism through elevated concentration and specific activity of cytochrome o

The function of the reversible oxygen-binding hemoprotein from Vitreoscilla (VHb), which enhances oxygen-limited cell growth and recombinant protein production when functionally expressed in Escherichia coli, was investigated in wild-type E. coli and in E. coli mutants lacking one of the two terminal oxidases, cytochrome o complex (aerobic terminal oxidase, Cyo) or cytochrome d complex (microaerobic terminal oxidase, Cyd). Deconvolution of VHb, cytochrome o, and cytochrome d bands from in vivo absorption spectra revealed a 5-fold enhancement in cytochrome o content and a 1.5-fold increment in cytochrome d by VHb under microaerobic environments (dissolved oxygen less than 2% air saturation). Based upon oxygen uptake kinetics measurements of these mutants, the apparent oxygen affinity of the Cyo(+), Cyd(-) E. coli was increased in the presence of VHb, but no difference in the apparent K(m) was observed for the Cyo(-), Cyd(+) strain. Results suggest that the expression of VHb in E. coli increases the level and activity of terminal oxidases and thereby improves the efficiency of microaerobic respiration and growth.     

Effect of Oxygen on Several Enzymes Involved in the Aerobic and Anaerobic Utilization of Glucose in Escherichia coli

By using the continuous culture technique, the transition from aerobiosis to anaerobiosis and its effect on a number of enzymes has been investigated in Escherichia coli K-12. A decrease in the oxygen partial pressure below 28.0 mm of Hg resulted firstly in an increase of the respiratory enzymes (reduced nicotinamide adenine dinucleotide [NADH] oxidase, 2.53-fold; succinic dehydrogenase, 1.4-fold; cytochrome b(1), 3.91-fold; and cytochrome a(2), 2.45-fold) before the electron transport system gradually collapsed as cytochrome a(2), followed by cytochrome b(1), succinic dehydrogenase, and finally NADH oxidase decreased in activity. The change from respiration to fermentation was initiated well before the oxygen tension reached zero by the increase in levels of fructose diphosphate-aldolase, glucose 6-phosphate, and 6-phosphogluconate dehydrogenases and a decrease in 2-oxoglutarate dehydrogenase. Whem the dissolved oxygen tension reached zero, dry weight and CO(2) formation together with isocitrate dehydrogenase decreased, whereas acid production and phosphofructokinase synthesis started to increase. Enzymatic investigations revealed that the kinetics of the enzyme phosphofructokinase from strict aerobic cultures (6.9 ppm oxygen in solution) was adenosine triphosphate (ATP)-insensitive, whereas the same enzyme from anaerobic cultures was ATP-sensitive. A mechanism is proposed for the change from aerobiosis to anaerobiosis together with the occurring change in glucose regulation.     

Intracellular expression of Vitreoscilla hemoglobin modifies microaerobic Escherichia coli metabolism through elevated concentration and specific activity of cytochrome o

The function of the reversible oxygen-binding hemoprotein from Vitreoscilla (VHb), which enhances oxygen-limited cell growth and recombinant protein production when functionally expressed in Escherichia coli, was investigated in wild-type E. coli and in E. coli mutants lacking one of the two terminal oxidases, cytochrome o complex (aerobic terminal oxidase, Cyo) or cytochrome d complex (microaerobic terminal oxidase, Cyd). Deconvolution of VHb, cytochrome o, and cytochrome d bands from in vivo absorption spectra revealed a 5-fold enhancement in cytochrome o content and a 1.5-fold increment in cytochrome d by VHb under microaerobic environments (dissolved oxygen less than 2% air saturation). Based upon oxygen uptake kinetics measurements of these mutants, the apparent oxygen affinity of the Cyo(+), Cyd(-) E. coli was increased in the presence of VHb, but no difference in the apparent K(m) was observed for the Cyo(-), Cyd(+) strain. Results suggest that the expression of VHb in E. coli increases the level and activity of terminal oxidases and thereby improves the efficiency of microaerobic respiration and growth. (c) 1996 John Wiley & Sons, Inc.     

A direct demonstration of "co-respiration" of oxygen and nitrogen oxides by Pseudomonas nautica: some spectral and kinetic properties of the respiratory components

Abstract: Pseudomonas nautica grown anaerobically is capable of simultaneously utilizing oxygen and nitrate or its reduced products (nitrite and nitrous oxide). Evidence for this 'co-respiration' came from kinetic studies on oxygen consumption depending on oxygen concentration and from spectral studies which revealed changes in the cytochromes composition of the electron transport chain under aerobic or anaerobic conditions. A constitutive o -type cytochrome oxidase was detected either aerobically or anaerobically with an apparent K _m for O₂ evaluated at 315 μM. Two oxidases were induced only in anaerobic conditions. One of these two enzymes identified as a cd -type cytochrome oxidase shows a relatively high affinity for oxygen with an apparent K _m value of 25 μM.     

Spectral evidence for the existence of a second cytochrome o in whole cells of Vitreoscilla

Cytochrome o, a protoheme IX pigment, has been proposed as the terminal oxidase of the filamentous bacterium, Vitreoscilla. Aerobic and anaerobic photolysis of CO-liganded whole cells demonstrated the presence of a second CO-reactive pigment, cytochrome o'. At temperatures lower than -100 degrees C, anaerobic photolysis dissociated only about 25% of the total CO-liganded components to reveal the unliganded cytochrome o'. At these temperatures, the photolysis of cytochrome o could not be demonstrated. At warmer temperatures, recombination of CO with the reduced cytochrome o' occurred with an apparent energy of activation of 5.8 kcal/mol. Aerobic photolysis of whole cells demonstrated two oxygen-bound intermediates. At temperatures lower than -95 degrees C, a spectrally distinct compound with absorption maxima at 428, 534, and 564 nm appeared (form I'); the apparent second order rate constant (k+1) for the formation of this intermediate was found to be 9.1 M-1 s-1, the reverse rate (k-1) was 9.9 X 10(-5) s-1, and the equilibrium constant (Kd) was 1.1 X 10(-5) M. This oxygen intermediate of cytochrome o' is spectrally and kinetically similar to the oxygen intermediate of cytochrome o seen in Escherichia coli. At temperatures warmer than -90 degrees C, photolysis of aerobic samples resulted in the immediate formation of a second oxygen-bound intermediate (form I) with absorption maxima at 422, 534, and 564 nm. This second intermediate results from the binding of oxygen to the cytochrome o (oxygenated cytochrome o). These data support the proposal that whole cells of Vitreoscilla contain two alternative pathways of electron transport, one terminating with cytochrome o and the other with cytochrome o'.     

Activities of respiratory enzymes during aerobic adaptation of anaerobically grownParacoccus denitrificans

The aerobic adaptation of anaerobically grownP. denitrificans carried out under conditions of limited growth is characterized by an exponential decrease of nitrite reductase activity and a sharp increase of cytochrome oxidase and a slow increase of NADH:cytochromec oxidase reductase and succinate dehydrogenase activities. The adaptation in a minimal adaptation medium under conditions of active or blocked protein synthesis showed that in addition to the degradation component of turnover during the aerobic adaptation other degradation enzyme(s), whose synthesis is induced by oxygen, are involved. This degradation system plays an essential role in the rapid disappearance of nitrite reductase and a pronounced decrease of the membranebound cytochromec oxidase activities during aerobic adaptation in the minimal adaptation medium.     

Localization and characterization of cytochromes from membrane vesicles of Escherichia coli K-12 grown in anaerobiosis with nitrate.

Cytochromes b of anaerobically nitrate-grown Escherichia coli cells are analysed. Ascorbate phenazine methosulfate distinguishes low and high potential cytochromes b. Reduction kinetics performed at 559 nm presents a very complex pattern which can be analysed assuming that at least four b-type cytochromes are present. The electron transport chain from formate to oxygen would contain a low potential cytochrome b-556, a cytochrome b-558 associated to the oxidase, and a cytochrome d as the principle oxidase. Cytochrome o is also present, but seems to be functional only at low oxygen concentrations. A cytochrome b-556 associated to nitrate reductase is shown to belong to a branch of the formate-oxidase chain. 2-N-Heptyl-4-hydroxyquinoline-N-oxide affects the reduction kinetics in a very complex way. One inhibition site is in evidence between cytochrome b-558 and cytochrome d; another between the cytochrome associated to nitrate reductase and the nitrate reductase. A third inhibition site is located in the common part of the formate-nitrate and the formate-oxidase systems. Ascorbate phenazine methosulfate is shown to donate electrons near cytochrome b-558.     

The sequence of the cyo operon indicates substantial structural similarities between the cytochrome o ubiquinol oxidase of Escherichia coli and the aa3-type family of cytochrome c oxidases

The cytochrome o complex is one of two ubiquinol oxidases in the aerobic respiratory system of Escherichia coli. This enzyme catalyzes the two-electron oxidation of ubiquinol-8 which is located in the cytoplasmic membrane, and the four-electron reduction of molecular oxygen to water. The purified oxidase contains at least four subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and has been shown to couple electron flux to the generation of a proton motive force across the membrane. In this paper, the DNA sequence of the cyo operon, containing the structural genes for the oxidase, is reported. This operon is shown to encode five open reading frames, cyoABCDE. The gene products of three of these, cyoA, cyoB, and cyoC, are clearly related to subunits II, I, and III, respectively, of the eukaryotic and prokaryotic aa3-type cytochrome c oxidases. This family of cytochrome c oxidases contain heme a and copper as prosthetic groups, whereas the E. coli enzyme contains heme b (protoheme IX) and copper. The most striking sequence similarities relate the large subunits (I) of both the E. coli quinol oxidase and the cytochrome c oxidases. It is likely that the sequence similarities reflect a common molecular architecture of the two heme binding sites and of a copper binding site in these enzymes. In addition, the cyoE open reading frame is closely related to a gene denoted ORF1 from Paracoccus dentrificans which is located in between the genes encoding subunits II and III of the cytochrome c oxidase of this organism. The function of the ORF1 gene product is not known. These sequence relationships define a superfamily of membrane-bound respiratory oxidases which share structural features but which have different functions. The E. coli cytochrome o complex oxidizes ubiquinol but has no ability to catalyze the oxidation of reduced cytochrome c. Nevertheless, it is clear that the E. coli oxidase and the aa3-type cytochrome c oxidases must have very similar structures, at least in the vicinity of the catalytic centers, and they are very likely to have similar mechanisms for bioenergetic coupling (proton pumping).     

Nitrate reductase and respiratory adaptation in Bacillus stearothermophilus

Downey, R. J. (University of Notre Dame, Notre Dame, Ind.). Nitrate reductase and respiratory adaptation in Bacillus stearothermophilus. J. Bacteriol. 91:634-641. 1966.-Bacillus stearothermophilus 2184 required nitrate to grow in the absence of oxygen. Like many facultative microorganisms, the growth obtained anaerobically was considerably less than that obtained aerobically, even though the dissimilatory reduction of nitrate is, in effect, anaerobic respiration. The ability to reduce nitrate depended on the induction of nitrate reductase. Although oxygen at low levels did not retard induction of the enzyme, enzyme synthesis was considerably lessened by aeration. A semisynthetic medium containing nitrate supported aerobic growth of the thermophile but did not support anaerobic growth. The adaptation to nitrate resulted in a decrease in the level of cytochrome oxidase normally present in aerobically grown cells. Although the aerobic oxidation of succinate by the respiratory enzymes from aerobically grown cells was inhibited by 2-N-heptyl-4-hydroxyquinoline-N-oxide, the anaerobic oxidation of succinate by nitrate in a similar preparation from nitrate-adapted cells was not. The nitrate reductase in the bacillus was strongly inhibited by cyanide and azide but not by carbon monoxide. The nitrate reductase catalyzed the anaerobic oxidation of reduced nicotinamide adenine dinucleotide, and appeared to transfer electrons from cytochrome b(1) to nitrate. Cytochrome c(1) did not appear to be involved in the transfer.     

Stopped-flow and rapid-scan studies of the redox behavior of cytochrome aco from facultative alkalophilic Bacillus

Cytochrome aco purified from an alkalophilic bacterium grown at pH 10 contains hemes a, b, and c as prosthetic groups, and their redox behavior was examined by using stopped-flow and rapid-scan techniques. Under anaerobic conditions the reduction of both heme a and c moieties with dithionite proceeded exponentially but with different rates, usually the former being reduced about 4 times faster than the latter. The reduction of protoheme was much slower, and a time-difference spectrum for this species was of a high spin type with absorption peaks at 433, 557, and 609 nm. Only the protoheme combined with CO, fulfilling the criteria for cytochrome o. Potentiometric titrations determined a midpoint potential of c heme to be 95 mV at pH 7.0 and 25 degrees C and suggested the presence of two forms of a heme with midpoint potentials of 250 and 323 mV. Cytochrome aco utilizes ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) to reduce oxygen relatively rapidly without added cytochrome c (Qureshi, M. H., Yumoto, I., Fujiwara, T., Fukumori, Y., Yamanaka, T. (1990) J. Biochem. 107, 480-485). During the steady state, however, heme a stayed almost fully reduced in contrast to a partial reduction of heme c. Even after exhaustion of the dissolved oxygen the extent of reduction of heme c was 60-70% that attained by the dithionite reduction. When ascorbate plus TMPD-reduced cytochrome aco was exposed to oxygen the reduced heme c was oxidized rapidly whereas the oxidation of reduced a heme was negligibly slow. The full reduction of heme a during the steady state and its extremely slow oxidation rendered participation of heme a in the oxidase reaction less likely. A novel peak appearing transiently around 567 nm during the reaction was tentatively ascribed to an intermediate form of protoheme, or o heme, which was thus supposed to react directly with molecular oxygen. These results suggest strongly that the main electron transfer pathway would be c----o----oxygen. A possible role of a in regulating the electron flow through the main pathway and its functional relationship to a heme in the aa3-type cytochrome oxidase were discussed.