Role Of Cytochrome Research Articles

Contribution of cytochrome P450 4A isoforms to renal functional response to inhibition of nitric oxide production in the rat.

20-Hydroxyeicosatetraenoic acid (20-HETE), a major renal eicosanoid, regulates renal function and contributes to renal responses following withdrawal of nitric oxide (NO). However, the role of 20-HETE-synthesizing isoforms in renal function resulting from NO inhibition is unknown. The present study evaluated the role of cytochrome (CYP)4A1, -4A2 and -4A3 isoforms on renal function in the presence and absence of NO. Antisense oligonucleotides (ASODN) to CYP4A1, -4A2 and -4A3 reduced 20-HETE synthesis and downregulated the expression of CYP4A isoforms in renal microsomes. Nomega-L-nitromethyl arginine ester (L-NAME, 25 mg kg(-1)), an inhibitor of NO production, increased mean arterial blood pressure (MABP, Delta = +18 to 26 mmHg), reduced renal blood flow (RBF, Delta = -1.8 to 2.9 ml min(-1)), increased renal vascular resistance (RVR, Delta = +47 to 54 mmHg ml(-1) min(-1)), reduced glomerular filtration rate (GFR), but increased sodium excretion (UNaV). ASODN to CYP4A1 and -4A2 but not -4A3 reduced basal MABP and RVR and increased basal GFR, while ASODN to CYP4A2 significantly reduced basal UNaV suggesting a differential role for CYP4A isoforms in the regulation of renal function. ASODN to CYP4A2 but not -4A1 or -4A3 blunted the increase in MABP by L-NAME (38 +/- 9 %, P < 0.05). ASODN to CYP4A1, -4A2 and -4A3 attenuated the reduction in RBF and the consequent increase in RVR by L-NAME with a potency order of CYP4A2 = CYP4A1 > CYP4A3. ASODN to CYP4A1 and -4A2 but not -4A3 attenuated L-NAME-induced reduction in GFR, but ASODN to all three CYP4A isoforms blunted the L-NAME-induced increase in UNaV (CYP4A3 > CYP4A1 >> CYP4A2). We conclude from these data that CYP4A isoforms contribute to different extents to basal renal function. Moreover, CYP4A2 contributes greatest to haemodynamic responses while CYP4A3 contributes greatest to tubular responses following NO inhibition. We therefore propose that NO differentially regulates the function of CYP4A1, -4A2, and -4A3 isoforms in the renal vasculature and the nephron.

Mitochondrial intermembrane proteins in cell death

Apoptosis is a form of programmed cell death important in the development and tissue homeostasis of multicellular organisms. Mitochondria have, next to their function in respiration, an important role in the apoptotic-signaling pathway. Malfunctioning at any level of the cell is eventually translated in the release of apoptogenic factors from the mitochondrial intermembrane space resulting in the organized demise of the cell. Some of these factors, such as AIF and endonuclease G, appear to be highly conserved during evolution. Other factors, like cytochrome c, have gained their apoptogenic function later during evolution. In this review, we focus on the role of cytochrome c, AIF, endonuclease G, Smac/DIABLO, Omi/HtrA2, Acyl-CoA-binding protein, and polypyrimidine tract-binding protein in the initiation and modulation of cell death in different model organisms. These mitochondrial factors may contribute to both caspase-dependent and caspase-independent processes in apoptotic cell death.

The role of cytochrome c oxidase deficient hippocampal neurones in Alzheimer's disease.

Defects of mitochondrial function have been proposed as a potential mechanism in the development and pathogenesis of Alzheimer's disease (AD) and neuronal apoptosis. Mitochondrial enzyme-deficient pyramidal neurones are found in greater quantities in the hippocampus of AD patients than in age-matched controls. The presence of these neurones indicates that high levels of mutant mtDNA (mitochondrial DNA), sufficient to cause a biochemical deficiency within individual neurones, occur more frequently in AD than in normal ageing. This study analyses the relationship of cytochrome c oxidase (COX)-deficient neurones with the neuropathological markers of AD, neurofibrillary tangles (NFTs) and amyloid plaques, as well as markers of neuronal apoptosis known to occur in AD brains. Frozen sections of hippocampi from three AD patients were used to directly colocalize in situ the presence of histochemically COX-deficient neurones with immunohistology for the classical neuropathological markers of AD, tau and beta-amyloid. In addition, we also directly colocalized these mitochondrial-enzyme deficient neurones using terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling and cleaved caspase-3. The distribution of amyloid plaques is anatomically distinct from the COX-deficient hippocampal pyramidal neurones and the neurones that contained NFTs or apoptotic labelling were always COX-positive. COX-deficient, succinate dehydrogenase-positive hippocampal neurones indicative of high mtDNA mutation load do not appear to be prone to apoptosis or to directly participate in the over production of tau or beta-amyloid. Biochemically significant mitochondrial defects do occur in AD and are likely to contribute to the overall central nervous system dysfunction in impairing neuronal function and possibly causing neurodegeneration via mechanisms other than apoptosis.

The role of cytochrome c in caspase activation in Drosophila melanogaster cells

The release of cytochrome c from mitochondria is necessary for the formation of the Apaf-1 apoptosome and subsequent activation of caspase-9 in mammalian cells. However, the role of cytochrome c in caspase activation in Drosophila cells is not well understood. We demonstrate here that cytochrome c remains associated with mitochondria during apoptosis of Drosophila cells and that the initiator caspase DRONC and effector caspase DRICE are activated after various death stimuli without any significant release of cytochrome c in the cytosol. Ectopic expression of the proapoptotic Bcl-2 protein, DEBCL, also fails to show any cytochrome c release from mitochondria. A significant proportion of cellular DRONC and DRICE appears to localize near mitochondria, suggesting that an apoptosome may form in the vicinity of mitochondria in the absence of cytochrome c release. In vitro, DRONC was recruited to a >700-kD complex, similar to the mammalian apoptosome in cell extracts supplemented with cytochrome c and dATP. These results suggest that caspase activation in insects follows a more primitive mechanism that may be the precursor to the caspase activation pathways in mammals.

Reduction of technetium(VII) by Desulfovibrio fructosovorans is mediated by the nickel-iron hydrogenase.

Resting cells of the sulfate-reducing bacterium Desulfovibrio fructosovorans grown in the absence of sulfate had a very high Tc(VII)-reducing activity, which led to the formation of an insoluble black precipitate. The involvement of a periplasmic hydrogenase in Tc(VII) reduction was indicated (i) by the requirement for hydrogen as an electron donor, (ii) by the tolerance of this activity to oxygen, and (iii) by the inhibition of this activity by Cu(II). Moreover, a mutant carrying a deletion in the nickel-iron hydrogenase operon showed a dramatic decrease in the rate of Tc(VII) reduction. The restoration of Tc(VII) reduction by complementation of this mutation with nickel-iron hydrogenase genes demonstrated the specific involvement of the periplasmic nickel-iron hydrogenase in the mechanism in vivo. The Tc(VII)-reducing activity was also observed with cell extracts in the presence of hydrogen. Under these conditions, Tc(VII) was reduced enzymatically to soluble Tc(V) or precipitated to an insoluble black precipitate, depending on the chemical nature of the buffer used. The purified nickel-iron hydrogenase performed Tc(VII) reduction and precipitation at high rates. These series of genetic and biochemical approaches demonstrated that the periplasmic nickel-iron hydrogenase of sulfate-reducing bacteria functions as a Tc(VII) reductase. The role of cytochrome c(3) in the mechanism is also discussed.

The presence of 9- cis-β-carotene in cytochrome b6f complex from spinach

Cytochrome b 6 f complex with stoichiometrically bound β-carotene molecule was purified from spinach chloroplasts. The configuration of this β-carotene was studied by reversed-phase HPLC and resonance Raman spectroscopy. Both the absorption spectrum of this β-carotene in dissociated state and the Raman spectrum in native state can be unambiguously assigned to a 9- cis configuration. This finding is in contrast to the predominantly all- trans isomers commonly found in membranes and protein–pigment complexes of chloroplasts, suggesting that the 9- cis-β-carotene is an authentic component and may have a unique structural and functional role in cytochrome b 6 f complex.

Photosynthetic Water Oxidation in Cytochromeb559 Mutants Containing a Disrupted Heme-binding Pocket

The role of cytochrome b(559) in photosynthetic oxygen evolution has been investigated in three chloroplast mutants of Chlamydomonas reinhardtii, in which one of the two histidine axial ligands to the heme, provided by the alpha subunit, has been replaced by the residues methionine, tyrosine, and glutamine. Photosystem two complexes functional for oxygen evolution could be assembled in the methionine and tyrosine mutants up to approximately 15% of wild type levels, whereas no complexes with oxygen evolution activity could be detected in the glutamine mutant. PSII supercomplexes isolated from the tyrosine and methionine mutants were as active as wild type in terms of light-saturated rates of oxygen evolution but in contrast to wild type contained no bound heme despite the presence of the alpha subunit. Oxygen evolution in the tyrosine and methionine mutants was, however, more sensitive to photoinactivation than the WT. Overall, these data establish unambiguously that a redox role for the heme of cytochrome b(559) is not required for photosynthetic oxygen evolution. Instead, our data provide new evidence of a role for cytochrome b(559) in the protection of the photosystem two complex in vivo.

Enzymatic removal of nitric oxide catalyzed by cytochrome c' in Rhodobacter capsulatus.

Cytochrome c' from Rhodobacter capsulatus has been shown to confer resistance to nitric oxide (NO). In this study, we demonstrated that the amount of cytochrome c' synthesized for buffering of NO is insufficient to account for the resistance to NO but that the cytochrome-dependent resistance mechanism involves the catalytic breakdown of NO, under aerobic and anaerobic conditions. Even under aerobic conditions, the NO removal is independent of molecular oxygen, suggesting cytochrome c' is a NO reductase. Indeed, we have measured the product of NO breakdown to be nitrous oxide (N(2)O), thus showing that cytochrome c' is behaving as a NO reductase. The increased resistance to NO conferred by cytochrome c' is distinct from the NO reductase pathway that is involved in denitrification. Cytochrome c' is not required for denitrification, but it has a role in the removal of externally supplied NO. Cytochrome c' synthesis occurs aerobically and anaerobically but is partly repressed under denitrifying growth conditions when other NO removal systems are operative. The inhibition of respiratory oxidase activity of R. capsulatus by NO suggests that one role for cytochrome c' is to maintain oxidase activity when both NO and O(2) are present.

The Escherichia coli CcmG protein fulfils a specific role in cytochrome c assembly.

In Escherichia coli K-12, c-type cytochromes are synthesized only during anaerobic growth with trimethylamine-N-oxide, nitrite or low concentrations of nitrate as the terminal electron acceptor. A thioredoxin-like protein, CcmG, is one of 12 proteins required for their assembly in the periplasm. Its postulated function is to reduce disulphide bonds formed between correctly paired cysteine residues in the cytochrome c apoproteins prior to haem attachment by CcmF and CcmH. We report that loss of CcmG synthesis by mutation was not compensated by a second mutation in disulphide-bond-forming proteins, DsbA or DsbB, or by the chemical reductant, 2-mercaptoethanesulphonic acid. An anti-CcmG polyclonal antibody was used in Western-blot analysis to probe the redox state of CcmG in mutants defective in the synthesis of other proteins essential for cytochrome c assembly. The oxidized form of CcmG accumulated not only in trxA or dipZ mutants defective in the transfer of electrons from the cytoplasm for disulphide isomerization and reduction reactions in the periplasm, but also in ccmF and ccmH mutants. The requirement of both CcmF and CcmH for the reduction of the disulphide bond in CcmG indicates that CcmG functions later than CcmF and CcmH in cytochrome c assembly, rather than in electron transfer from the membrane-associated DipZ (also known as DsbD) to CcmH. The data support a model proposed by others in which CcmG catalyses one of the last reactions specific to cytochrome c assembly.

Cytochrome b5 plays a key role in human microsomal chromium(VI) reduction

The reduction of chromium(VI) to Cr(III) results in the formation of reactive intermediates that contribute to the cytotoxicity, genotoxicity, and carcinogenicity of Cr(VI)-containing compounds. Previous studies suggest that human microsomal Cr(VI) reduction likely proceeds through cytochrome b 5 . In order to better understand Cr(VI) toxicity in humans, the role of cytochrome b 5 in combination with P450 reductase was examined in the reductive transformation of Cr(VI). Proteoliposomes containing human recombinant cytochrome b 5 and P450 reductase were constructed. The ability of P450 reductase to mediate efficient electron transfer from NADPH to cytochrome b 5 was confirmed by spectral analysis. The NADPH-dependent Cr(VI) reduction rate mediated by proteoliposomes was then compared to that of human microsomes. When these rates were normalized to equivalent cytochrome b 5 concentrations, the NADPH-dependent Cr(VI) reduction rates mediated by human microsomes were essentially identical to those for proteoliposomes containing cytochrome b 5 plus P450 reductase. Proteoliposomes containing only P450 reductase or cytochrome b 5 exhibited poor Cr(VI) reducing capabilities. Since it had been previously shown that trace amounts of iron (Fe) could dramatically stimulate microsomal Cr(VI) reduction, the ability of Fe to stimulate Cr(VI) reduction by proteoliposomes was examined. Both ferric chloride (FeCl 3) and ferric adenosine-5′-diphosphate (FeADP) were shown to stimulate Cr(VI) reduction; this stimulation could be abolished by the addition of deferoxamine, a specific Fe(III) chelator. The NADPH-dependent reduction rates of various ferric complexes by proteoliposomes were sufficient to account for the increased Cr(VI) reduction rates seen with the addition of FeCl 3 or FeADP. Cr(V) was detected by electron paramagnetic resonance (EPR) spectroscopy as a transient intermediate formed during NADPH-dependent Cr(VI) reduction mediated by proteoliposomes containing cytochrome b 5 and P450 reductase. Overall, cytochrome b 5 in combination with P450 reductase can account for the majority of the NADPH-dependent Cr(VI) reduction seen with human microsomes.

An absolutely conserved tryptophan: investigating its role in cytochrome c

An absolutely conserved tryptophan: investigating its role in cytochrome c

Eosinophil apoptosis--pathophysiologic and therapeutic implications.

Eosinophil apoptosis--pathophysiologic and therapeutic implications.

Apamin-sensitive, non-nitric oxide (NO) endothelium-dependent relaxations to bradykinin in the bovine isolated coronary artery: no role for cytochrome P450 and K+.

Since cytochrome P(450)-derived metabolites of arachidonic acid and K(+) have been implicated in endothelium-derived hyperpolarizing factor (EDHF)-dependent responses, the aim of this study was to determine whether such factors contribute to non-nitric oxide (NO), endothelium-dependent relaxation to bradykinin (BK) in bovine isolated coronary artery. In rings of artery contracted with U46619 and treated with indomethacin (3 microM) and N(G)-nitro-L-arginine (L-NOARG; 100 microM), relaxation to BK (0.01 nM-0.3 microM) was blocked by approximately 60% after inhibition of K(+) channels with either high extracellular K(+) (high [K(+)](o); 15 - 67 mM) or apamin (0.3 microM). Ouabain (1 microM), an inhibitor of Na(+)/K(+)-ATPase, decreased the sensitivity to BK without affecting the maximum response. In L-NOARG-treated rings, ouabain had no further effect on the relaxation to BK. An inhibitor of inward-rectifying K(+) channels, Ba(2+) (30 microM), had no effect on relaxations to BK in the absence or presence of either L-NOARG or ouabain. KCl (2.5 - 10 mM) elicited small relaxations ( approximately 20%) that were abolished by nifedipine (0.3 microM) and ouabain. Both the high [K(+)](o)/apamin-sensitive relaxation to BK, and the relaxation to the K(ATP) channel-opener, levcromakalim (0.6 microM), were unaffected by the cytochrome P(450) inhibitor, 7-ethoxyresorufin (10 microM), or by co-treatment with a phospholipase A(2) inhibitor, arachidonyl trifluoromethyl ketone (AACOCF(3); 3 microM) and a diacylglycerol (DAG)-lipase inhibitor, 1, 6-bis-(cyclohexyloximinocarbonylamino)-hexane (RHC 80267; 30 microM). The non-NO/high [K(+)](o)-insensitive, approximately 40% relaxation to BK was, however, abolished by these treatments. Therefore, neither cytochrome P(450)-derived metabolites of arachidonic acid nor K(+) appear to mediate the EDHF-like relaxation to BK (i.e the non-NO, high [K(+)](o)/apamin-sensitive component) in bovine coronary arteries. Cytochrome P(450)-derived metabolites may be released at higher BK concentrations to act in parallel with NO and the high [K(+)](o)/apamin-sensitive mechanism.

The antioxidant functions of cytochrome c

Low (C 1/2=1.5×10 −7 M) concentrations of horse cytochrome c strongly inhibit H 2O 2 production by rat heart mitochondria under conditions of reverse electron transfer from succinate to NAD +. The effect is abolished by binding of cytochrome c with liposomes and is not prevented by SOD. Yeast cytochrome c is much less effective than the horse protein whereas acetylated horse cytochrome c is without effect. H 2O 2 formation stimulated by antimycin A is resistant to added cytochrome c. In inside-out submitochondrial vesicles, H 2O 2 production is suppressed by all three cytochrome c samples tested, but at higher concentrations (C 1/2 is about 5×10 −7 M). In vesicles, SOD abolishes the cytochrome c inhibition. We conclude that extramitochondrial cytochrome c is competent in down-regulation of the Complex I H 2O 2 production linked to the reverse electron transfer. Such an effect is absent in the inside-out submitochondrial vesicles where another antioxidant cytochrome c function can be observed, i.e. the oxidation of O 2 − to O 2. A possible role of cytochrome c in the antioxidant defence is discussed.

Role of Cytochrome c as a Stimulator of α-Synuclein Aggregation in Lewy Body Disease

alpha-Synuclein is a major component of aggregates forming amyloid-like fibrils in diseases with Lewy bodies and other neurodegenerative disorders, yet the mechanism by which alpha-synuclein is intracellularly aggregated during neurodegeneration is poorly understood. Recent studies suggest that oxidative stress reactions might contribute to abnormal aggregation of this molecule. In this context, the main objective of the present study was to determine the potential role of the heme protein cytochrome c in alpha-synuclein aggregation. When recombinant alpha-synuclein was coincubated with cytochrome c/hydrogen peroxide, alpha-synuclein was concomitantly induced to be aggregated. This process was blocked by antioxidant agents such as N-acetyl-L-cysteine. Hemin/hydrogen peroxide similarly induced aggregation of alpha-synuclein, and both cytochrome c/hydrogen peroxide- and hemin/hydrogen peroxide-induced aggregation of alpha-synuclein was partially inhibited by treatment with iron chelator deferoxisamine. This indicates that iron-catalyzed oxidative reaction mediated by cytochrome c/hydrogen peroxide might be critically involved in promoting alpha-synuclein aggregation. Furthermore, double labeling studies for cytochrome c/alpha-synuclein showed that they were colocalized in Lewy bodies of patients with Parkinson's disease. Taken together, these results suggest that cytochrome c, a well known electron transfer, and mediator of apoptotic cell death may be involved in the oxidative stress-induced aggregation of alpha-synuclein in Parkinson's disease and related disorders.

Apoptosis, the Role of Oxidative Stress and the Example of Solar UV Radiation

Apoptosis, the Role of Oxidative Stress and the Example of Solar UV Radiation

Role of cytochrome c and dATP/ATP hydrolysis in Apaf-1-mediated caspase-9 activation and apoptosis.

Apaf-1 plays a critical role in apoptosis by binding to and activating procaspase-9. We have identified a novel Apaf-1 cDNA encoding a protein of 1248 amino acids containing an insertion of 11 residues between the CARD and ATPase domains, and another 43 amino acid insertion creating an additional WD-40 repeat. The product of this Apaf-1 cDNA activated procaspase-9 in a cytochrome c and dATP/ATP-dependent manner. We used this Apaf-1 to show that Apaf-1 requires dATP/ATP hydrolysis to interact with cytochrome c, self-associate and bind to procaspase-9. A P-loop mutant (Apaf-1K160R) was unable to associate with Apaf-1 or bind to procaspase-9. Mutation of Met368 to Leu enabled Apaf-1 to self-associate and bind procaspase-9 independent of cytochrome c, though still requiring dATP/ATP for these activities. The Apaf-1M368L mutant exhibited greater ability to induce apoptosis compared with the wild-type Apaf-1. We also show that procaspase-9 can recruit procaspase-3 to the Apaf-1-procaspase-9 complex. Apaf-1(1-570), a mutant lacking the WD-40 repeats, associated with and activated procaspase-9, but failed to recruit procaspase-3 and induce apoptosis. These results suggest that the WD-40 repeats may be involved in procaspase-9-mediated procaspase-3 recruitment. These studies elucidate biochemical steps required for Apaf-1 to activate procaspase-9 and induce apoptosis.

Functional response of the rat kidney to inhibition of nitric oxide synthesis: role of cytochrome p450-derived arachidonate metabolites.

1. We tested the hypothesis that nitric oxide (NO) exerts a tonic inhibitory influence on cytochrome P450 (CYP450)-dependent metabolism of arachidonic acid (AA). 2. N(omega)-nitro-L-Arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase (NOS), increased mean blood pressure (MBP), from 91+/-6 to 137+/-5 mmHg, renal vascular resistance (RVR), from 9.9+/-0.6 to 27.4+/-2.5 mmHg ml(-1) min(-1), and reduced renal blood flow (RBF), from 9.8+/-0.7 to 6.5+/-0.6 ml min(-1)) and GFR from 1.2+/-0.2 to 0.6+/-0.2 ml 100 g(-1) min(-1)) accompanied by diuresis (UV, 1.7+/-0.3 to 4.3+/-0.8 microl 100 g(-1) min (-1)), and natriuresis (U(Na)V, 0.36+/-0.04 to 1.25+/-0.032 micromol 100 g(-1) min(-1)). 3. 12, 12 dibromododec-enoic acid (DBDD), an inhibitor of omega hydroxylase, blunted L-NAME-induced changes in MBP, RVR, UV and U(Na)V by 63+/-8, 70+/-5, 45+/-8 and 42+/-9%, respectively, and fully reversed the reduction in GFR by L-NAME. Clotrimazole, an inhibitor of the epoxygenase pathway of CYP450-dependent AA metabolism, was without effect. 4. BMS182874 (5-dimethylamino)-N-(3,4-dimethyl-5-isoxazolyl)-1-naphthalenesulfo namide), an endothelin (ET)A receptor antagonist, also blunted the increases in MBP and RVR and the diuresis/natriuresis elicited by L-NAME without affecting GFR. 5. Indomethacin blunted L-NAME-induced increases in RVR, UV and U(Na)V. BMS180291 (1S-(1alpha,2alpha,3alpha,4alpha)]-2-[[3-[4-[(++ +pentylamino)carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl ]methyl]benzenepropanoic acid), an endoperoxide receptor antagonist, attenuated the pressor and renal haemodynamic but not the renal tubular effects of L-NAME. 6. In conclusion, the renal functional effects of the CYP450-derived mediator(s) expressed after inhibition of NOS with L-NAME were prevented by inhibiting either CYP450 omega hydroxylase or cyclooxygenase or by antagonizing either ET(A) or endoperoxide receptors. 20-hydroxyeicosatetraenoic acid (20-HETE) fulfils the salient properties of this mediator.

Mitochondrial control of apoptosis: the role of cytochrome c

Mitochondrial cytochrome c (cyt c) has been found to have dual functions in controlling both cellular energetic metabolism and apoptosis. Through interaction with apoptotic protease activating factors (Apaf), cyt c can initiate the activation cascade of caspases once it is released into the cytosol. The loss of a component of the mitochondrial electron transport chain also triggers the generation of superoxide. Although cyt c can be released independent of the mitochondrial permeability transition (MPT), the accompanying cellular redox change can trigger the MPT. Since another apoptotic protease, AIF, is released by MPT, the two separate pathways provide redundancy that ensures effective execution of the cell death program. Anti-apoptotic Bcl-2 family proteins function as gatekeepers to prevent the release of both cyt c and AIF. In spite of their stabilization effect on the mitochondrial outer membrane, Bcl-2 proteins may also be involved in the direct binding of Apaf molecules as regulatory elements further downstream from the mitochondrial apoptotic signals.

Paracoccus denitrificans cytochrome c oxidase: a kinetic study on the two- and four-subunit complexes

Cytochrome c oxidase from Paracoccus denitrificans has been purified in two different forms differing in polypeptide composition. An enzyme containing polypeptides I–IV is obtained when the purification procedure is performed in β- d-dodecylmaltoside. If, however, Triton X-100 is used to purify the enzyme under otherwise identical conditions, an enzyme is obtained containing only subunits I–II. The two enzymes are undistinguishable by optical spectroscopy but show significant differences in the transient and steady-state time regimes, as studied by stopped-flow spectroscopy. The observed differences, however, are not due to removal of subunits III and IV, but rather to a specific effect of Triton X-100 which appears to affect cytochrome c binding. From these results it is not expected that subunits III and IV play any significant role in cytochrome c binding and, possibly, in the subsequent electron transfer processes. The results also suggest that both electrostatic and hydrophobic interactions may be important in the initial electron transfer process from cytochrome c.