Changes in the spin state and reactivity of cytochrome C induced by photochemically generated singlet oxygen and free radicals

This work compares the effect of photogenerated singlet oxygen (O(2)((1)Delta(g))) (type II mechanism) and free radicals (type I mechanism) on cytochrome c structure and reactivity. Both reactive species were obtained by photoexcitation of methylene blue (MB(+)) in the monomer and dimer forms, respectively. The monomer form is predominant at low dye concentrations (up to 8 microm) or in the presence of an excess of SDS micelles, while dimers are predominant at 0.7 mm SDS. Over a pH range in which cytochrome c is in the native form, O(2) ((1)Delta(g)) and free radicals induced a Soret band blue shift (from 409 to 405 nm), predominantly. EPR measurements revealed that the blue shift of the Soret band was compatible with conversion of the heme iron from its native low spin state to a high spin state with axial symmetry (g approximately 6.0). Soret band bleaching, due to direct attack on the heme group, was only detected under conditions that favored free radical production (MB(+) dimer in SDS micelles) or in the presence of a less structured form of the protein (above pH 9.3). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry of the heme group and the polypeptide chain of cytochrome c with Soret band at 405 nm (cytc405) revealed no alterations in the mass of the cytc405 heme group but oxidative modifications on methionine (Met(65) and Met(80)) and tyrosine (Tyr(74)) residues. Damage of cytc405 tyrosine residue impaired its reduction by diphenylacetaldehyde, but not by beta-mercaptoethanol, which was able to reduce cytc405, generating cytochrome c Fe(II) in the high spin state (spin 2).     

Studies on the reaction of imidazole with cytochrome c3 from Desulfovibrio vulgaris

1. Cytochrome c₃, a unique hemoprotein with a negative redox potential and four heme groups bound to a single polypeptide chain, reacts with imidazole in the reduced state to form a low-spin ferro · imidazole complex which is spectrally characterized by a 3.1 nm blue shift in the α-peak (from 550.5 to 547.4 nm). The spectral imidazole · cytochrome c₃ complex is detectable at 77 but not at 298 K.2. Mammalian ferrocytochrome c did not undergo a spectral interaction with imidazole at either 77 or 298 K, indicating that the imidazole · cytochrome c₃ complex reflects a unique event for cytochrome c₃.3. Formation of the imidazole · cytochrome c₃ complex is strongly dependent on imidazole concentration (apparent K_d of approx. 50 mM), and is abolished in the presence of 100 mM phosphate. This latter effect is attributable to formation of an imidazole · phosphate complex. A pH titration of the imidazole · cytochrome c₃ spectral complex implicates ionization of an imidazole function (pK = 8.5).4. EPR studies at 8.5 K of ferricytochrome c₃ before and after one reduction-oxidation cycle indicate that at least two of the hemes undergo reaction with imidazole forming two different low-spin ferric heme · imidazole complexes, with significant shifts in the g values of two heme signals.5. The spectral and EPR results are consistent with formation as the primary event of a low-spin ferrocytochrome c₃ · imidazole complex in which increased hydrophobicity and protonation-deprotonation effects are contributary to the consequent lability of cytochrome c₃.     

The cytochrome c oxidase-cytochrome c complex: spectroscopic analysis of conformational changes in the protein-protein interaction domain

Binding to cytochrome c oxidase induces a conformational change in the cytochrome c molecule. This conformational change has been characterized by comparing the binding of native cytochrome c and chemically modified cytochrome c derivatives to bovine cytochrome c oxidase by using absorption, circular dichroism (CD), and magnetic circular dichroism (MCD) spectroscopy. The following derivatives were analyzed: (i) cytochrome c modified at all 19 lysine residues to yield the (N epsilon-acetimidyl)19 cytochrome c, (N epsilon-isopropyl)19 cytochrome c, and (N epsilon,N epsilon-dimethyl)19 cytochrome c; (ii) cytochrome c in which Met65 and Met80 are converted to the methionine sulfoxide; (iii) cytochrome c with a single break in the polypeptide chain at Arg38 or Gly37. The derivatives bind to cytochrome c oxidase at a ratio of one heme c per heme aa3. The association constants are similar to that of native cytochrome c except for (N epsilon-isopropyl)19 and (N epsilon,N epsilon-dimethyl)19 cytochromes c, which bind respectively four times and six times less strongly. The derivatives are good substrates for the cytochrome c oxidase reaction. The spectral changes accompanying the binding of the modified cytochromes c to cytochrome c oxidase are quite different from the spectral changes observed with native cytochrome c. The different optical absorption and MCD changes are explained by a polarity change around the exposed heme edge in the cytochrome c-cytochrome c oxidase complex. The CD changes indicate a conformational rearrangement restricted to the surface area surrounding the exposed heme edge. The rearrangement may involve a movement of the evolutionarily conserved Phe82 out of the vicinity of the heme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spectroscopic and oxidation-reduction properties of Rhodobacter capsulatus cytochrome c1 and its M183K and M183H variants

Two variants of the cytochrome c1 component of the Rhodobacter capsulatus cytochrome bc1 complex, in which Met183 (an axial heme ligand) was replaced by lysine (M183K) or histidine (M183H), have been analyzed. Electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) spectra of the intact complex indicate that the histidine/methionine heme ligation of the wild-type cytochrome is replaced by histidine/lysine ligation in M183K and histidine/histidine ligation in M183H. Variable amounts of histidine/histidine axial heme ligation were also detected in purified wild-type cytochrome c1 and its M183K variant, suggesting that a histidine outside the CSACH heme-binding domain can be recruited as an alternative ligand. Oxidation-reduction titrations of the heme in purified cytochrome c1 revealed multiple redox forms. Titrations of the purified cytochrome carried out in the oxidative or reductive direction differ. In contrast, titrations of cytochrome c1 in the intact bc1 complex and in a subcomplex missing the Rieske iron-sulfur protein were fully reversible. An Em7 value of -330 mV was measured for the single disulfide bond in cytochrome c1. The origins of heme redox heterogeneity, and of the differences between reductive and oxidative heme titrations, are discussed in terms of conformational changes and the role of the disulfide in maintaining the native structure of cytochrome c1.     

Methionine sulfoxide cytochrome c.

Cytochrome c has been chemically modified by methylene blue mediated photooxidation. It is established that the methionine residues of the protein have been specifically converted to methionine sulfoxide residues. No oxidation of any other amino acid residues or the cysteine thioether bridges of the molecule occurs during the photooxidation reaction. The absorbance spectrum of methionine sulfoxide ferricytochrome c at neutrality is similar to that of the unmodified protein except for an increase in the extinction coefficient of the Soret absorbance band and for the complete loss of the ligand sensitive 695 nm absorbance band in the spectrum of the derivative. The protein remains in the low spin configuration which implies the retention of two strong field ligands. Spin state sensitive spectral titrations and model studies of heme peptides indicate that the sixth ligand is definitely not provided by a lysine residue but may be methionine-80 sulfoxide coordinated via its sulfur atom. Circular dichroism spectra indicate that the heme crevice of methionine sulfoxide ferri- and ferrocytochrome c is weakened relative to native cytochrome c. The redox potential of methionine sulfoxide cytochrome c is 184 mV which is markedly diminished from the 260 mV redox potential of native cytochrome c. The modified protein is equivalent to native cytochrome c as a substrate for cytochrome oxidase and is not autoxidizable at neutral pH but is virtually inactive with succinate-cytochrome c reductase. These results indicate that the major role of the methionine-80 in cytochrome c is to preserve a closed hydrophobic heme crevice which is essential for the maintainance of the necessary redox potential.     

Kinetic intermediates in the formation of ordered complexes from cytochrome c fragments. Evidence that methionine ligation is a late event in the folding process

The reactions of ferric heme-containing fragments with apofragments to form ordered complexes resembling native horse heart cytochrome c have been studied under conditions which resolve the overall process into consecutive second order and first order kinetic steps. In the initial, second order step the two fragments combine to form an intermediate complex which exhibits tryptophan 59 fluorescence quenching similar to native cytochrome c, but which has not yet achieved the native ligation state of the heme iron. The existence of first order processes following the second order step is demonstrated by absorbance changes in the Soret region. the entire absorbance change at 695 nm, relating to ligation of the sulfur atom of methionine 80 to the heme iron, is also associated with these first order processes. Thus, ligation of methionine is a late event in this self ordering of the polypeptide chains. Since the conformational energy is assumed to distinctly decrease in this late process of folding (Parr, G.R., and Taniuchi, H. (1980) J. Biol. Chem. 255, 2616-2623), it would follow that small spatial rearrangements of the polypeptide chains in the late stage of folding (as manifested by the ligation of methionine) are associated with a specific decrease in energy.     

Some properties of mammalian cardiac cytochrome c1.

Investigations into the nature of the axial heme ligands, the strength of the heme crevice, the reactivity with cyanide, and the ascorbate reducibility of cytochrome c1 were performed to explore structure-function relationships of cytochrome c1. The existence of an absorbance band at 690 nm, which was quenched by raising the pH with a pK of 9.2 corresponding to a low spin-low transition, suggested that a methionine residue probably functioned as one of the axial heme iron ligands in this cytochrome. Spectral titrations of cytochrome c1 in the low pH range showed a markedly elevated pK for the low spin-high spin transition relative to cytochrome c. Denaturation studies with urea, the absence of any reaction with cyanide, and the evidence from other lines would appear to indicate that the heme group of cytochrome c1 was reduced by ascorbate at approximately 5% of the rate of reduction of cytochrome c but this rate dramatically increased with increasing pH concomitant with the disappearance of the 690 nm absorbance band. Circular dichroic spectra substantiated that elevated pH produced conformational changes localized to the heme crevice and probably also the regions containing aromatic residues. The enhanced rate of ascorbate reduction was perhaps a consequence of the increased accessibility of the heme iron to ascorbate. Major unfolding of the protein in 8 M urea, however, completely abolished the ascorbate reducibility of cytochrome c1. The buried nature of the heme group of cytochrome c1 would probably preclude transfer of an electron from cytochrome c1 to cytochrome c through a direct Fe-Fe or a heme-heme interaction. This poses an important question concerning the mechanism of this electron transfer between these two cytochromes not only in mitochondria but also in solution.     

Axial ligation and heme environment in cytochrome c-555 from Prosthecochloris aestuarii. Investigation by absorption and solvent perturbation difference spectroscopy.

The near-IR absorption spectrum indicated that methionine is the sixth axial heme iron ligand in Prosthecochloris aestuarii cytochrome c-555. The heme environment has been investigated by the technique of solvent perturbation difference spectroscopy. The heme octapeptide from cytochrome c plus added imidazole was used as a model compound for the fully exposed chromophore. The heme was found to be minimally exposed to solvent. A comparison was made with cytochrome c, as to the possible causes of the difference in redox potentials betweeen these two cytochromes.     

Cleavage of the iron-methionine bond in c-type cytochromes: Crystal structure of oxidized and reduced cytochrome c2 from Rhodopseudomonas palustris and its ammonia complex

The three-dimensional structures of the native cytochrome c(2) from Rhodopseudomonas palustris and of its ammonia complex have been obtained at pH 4.4 and pH 8.5, respectively. The structure of the native form has been refined in the oxidized state at 1.70 A and in the reduced state at 1.95 A resolution. These are the first high-resolution crystal structures in both oxidation states of a cytochrome c(2) with relatively high redox potential (+350 mV). The differences between the two oxidation states of the native form, including the position of internal water molecules, are small. The unusual six-residue insertion Gly82-Ala87, which precedes the heme binding Met93, forms an isolated 3(10)-helix secondary structural element not previously observed in other c-type cytochromes. Furthermore, this cytochrome shows an external methionine residue involved in a strained folding near the exposed edge of the heme. The structural comparison of the present cytochrome c(2) with other c-type cytochromes has revealed that the presence of such a residue, with torsion angles phi and psi of approximately -140 and -130 degrees, respectively, is a typical feature of this family of proteins. The refined crystal structure of the ammonia complex, obtained at 1.15 A resolution, shows that the sulphur atom of the Met93 axial ligand does not coordinate the heme iron atom, but is replaced by an exogenous ammonia molecule. This is the only example so far reported of an X-ray structure with the heme iron coordinated by an ammonia molecule. The detachment of Met93 is accompanied by a very localized change in backbone conformation, involving mainly the residues Lys92, Met93, and Thr94. Previous studies under typical denaturing conditions, including high-pH values and the presence of exogenous ligands, have shown that the detachment of the Met axial ligand is a basic step in the folding/unfolding process of c-type cytochromes. The ammonia adduct represents a structural model for this important step of the unfolding pathway. Factors proposed to be important for the methionine dissociation are the strength of the H-bond between the Met93 and Tyr66 residues that stabilizes the native form, and the presence in this bacterial cytochrome c(2) of the rare six-residue insertion in the helix 3(10) conformation that increases Met loop flexibility.     

Purification and properties of cytochrome c-553, an electron acceptor for formate dehydrogenase of Desulfovibrio vulgaris, Miyazaki

Cytochrome c-553 of Desulfovibrio vulgaris, Miyazaki, was purified to homogeneity. The absorption spectrum of the ferro form has four peaks at 553, 525, 417 and 317 nm with a plateau near 280 nm, and that of the ferri form has three peaks at 525, 410 and 360 nm with a plateau near 280 nm and a shoulder at 560 nm. The millimolar absorbance coefficient of the α-peak of the ferro form is 23.9. The molecular weight of cytochrome c-553 is 8000, and it contains one heme. Its isoelectric point is rather alkaline, and its standard redox potential is ?0.26 V at pH 7.0. Its amino acid composition is unique; it lacks proline, isoleucine and tryptophan.Ferrocytochrome c-553 does not combine with CO, nor does it transfer electrons directly to various redox carriers such as flavin nucleotides, methylene blue, indigodisulfonate, 5-methylphenazinium methyl sulfate, 1-methoxy-5-methylphenazinium methyl sulfate, viologens and cytochrome c₃, but is oxidized by ferricyanide or by O₂.Cytochrome c-553 can be reduced by formate dehydrogenase of this bacterium in the presence of formate, but not by hydrogenase under H₂. The formate dehydrogenase does not reduce cytochrome c₃ in the presence of formate. The systematic name for formate dehydrogenase of D. vulgaris is, therefore, established as formate:ferricytochrome c-553 oxidoreductase in EC subclass 1.2.2.—.     

Contents to volume 164

Cytochrome b₆ from spinach chloroplasts (either within the purified cytochrome b₆f complex, or in its isolated form) exhibits two spectral species, which correspond to two midpoint potentials. This can be demonstrated by low temperature difference spectroscopy at fixed redox potentials. The high potential form of cytochrome b₆ has a split α-peak at 557.5 and 561.5 nm, the low potential form has a symmetrical α-peak at 560.5 nm. Similar results were obtained with cytochrome b₆ in the isolated cytochrome b₆f complex from the cyanobacterium Anabaena variabilis.     

Purification and some properties of cytochrome C553(550) isolated from Desulfovibrio desulfuricans Norway.

A new c-type cytochrome containing a single heme group, cytochrome c_553(550) has been purified from $\text{Desulfovibrio desulfuricans}$ (Norway strain) and some of its properties have been investigated. It has an isoelectric point of 6.6 and a higher redox potential than cytochrome c₃ isolated from the same bacteria. Its molecular weight was estimated to be 9,200 by gel filtration. The main absorption peaks are at 553, 522.5 and 417 nm in the reduced form and at 690, 529, 411, 357 and 280 nm in the oxidized form. The asymmetric α band of the reduced state is similar to the one reported for socalled “split α” cytochromes c. The cytochrome contains 86 amino acid residues with 5 methionine, two cysteine and two histidine residues. The N terminal sequence of $\text{D. desulfuricans}$ Norway cytochrome c_553(550) presents no evident homology with that of $\text{Desulfovibrio vulgaris}$ Hildenborough cytochrome c₅₅₃.     

The interaction between heme and protein in cytochrome c1

The optical spectrum of reduced bovine cytochrome c₁ at 77 K shows a fine splitting of the β-band, which is indicative of the native conformation of the protein. At room temperature, this conformation is reflected in an absorbance band at 530 nm. The exposure of the heme of ferrocytochrome c₁, investigated by means of solvent-perturbation spectroscopy, appears to be extremely sensitive to temperature and SH reagents bound to the oxidized protein. Addition of combinations of potential ligands to the isolated tryptic heme peptide of cytochrome c₁ reveals that only a mixture of methionine and cysteine (or their equivalents) generates a β-band at 77 K which is identical in shape to that of native cytochrome c₁. In the EPR spectrum of a complex of ferrocytochrome c₁ and nitric oxide at pH 10.5, no hyperfine splitting derived from a second ligated nitrogen atom could be detected. The results indicate that methionine and cysteine are the axial ligands of heme in cytochrome c₁. The EPR spectrum of isolated ferricytochrome c₁ is that of a low-spin heme iron compound with a g_z value of 3.36 and a g_y value of 2.04.     

Partial purification and characterization of two soluble c-type cytochromes from Chromatium vinosum

Two c-type cytochromes from Chromatium vinosum have been partially purified and characterized. Cytochrome c₅₅₀, which appears to function as an electron carrier in the cyclic electron transport chain of this photosynthetic purple sulfur bacterium, has a molecular weight of approximately 15,000 and an oxidation-reduction midpoint potential (E_m) of + 240 mV at pH 7.4. It has (in the reduced form) an α band at 550 nm and a β band at 520 nm. Cytochrome c₅₅₁ is characterized by absorbance maxima at 354 and 409 nm in the oxidized form and 418, 523, and 551 nm in the reduced form. The reduced cytochrome reacts with CO. Cytochrome c₅₅₁ is a monomeric protein with a molecular weight of 18,800 ± 700 and E_m = ?299 ± 5 mV (pH independent between pH 6.3 and 8.0). It appears to lack a methionine axial ligand as indicated by the absence of an absorbance band at 695 nm in the oxidized form.     

Kinetic Studies on the Interaction of Ferricytochrome c with Anionic Surfactants

The kinetics of absorbance and fluorescence changes of cytochrome c as induced by an aqueous solution of the anionic surfactant sodium dodecyl sulfate (SDS) or sodium bis(2-ethylhexyl)sulfosuccinate (AOT) are studied. The results are compared with far-UV circular dichroism (CD) spectra. Both surfactants cause similar alterations in the secondary structure of cytochrome c, while their influence on the heme environment of cytochrome c is different. In the presence of AOT below and above critical micellar concentration a conversion of the low-spin native cytochrome c to a denatured low-spin protein not having methionine ligand takes place. In the presence of SDS micelles conversion of the native protein to a denatured mixed-spin form occurs. The changes in the heme group induced by both surfactants occur independently of the alterations in tertiary structure.     

Folding character of cytochrome c studied by o-nitrobenzyl modification of methionine 65 and subsequent ultraviolet light irradiation

The protein folding character of cyt c was studied with the use of a photocleavable o-nitrobenzyl derivative of Met65 (NBz-Met65). For the NBz-Met65 cyt c, the Soret absorption band slightly blue shifted compared with the unlabeled cyt c, the 695 nm absorption band related to the Met80 sulfur ligation to the heme iron disappeared, and its resonance Raman spectrum was characteristic of a six-coordinate low-spin species, all characters demonstrating coordination of a non-native ligand, probably a histidine, instead of Met80 to the heme iron. The far-UV circular dichroism (CD) spectrum of cyt c was altered, and the transition midpoint concentration value of guanidine hydrochloride (GdnHCl) for unfolding the protein decreased by 0.9 M by the modification, which showed perturbation of the structure and decrease in protein stability, respectively. With irradiation of 308 nm laser pulses on the NBz-Met65 cyt c, the Soret absorption band slightly red shifted, the 695 nm absorption band appeared, and the CD spectrum shifted toward that of the native protein, which demonstrated recovery of the methionine heme coordination and the native protein structure, due to reconversion of NBz-Met65 to unlabeled methionine. A fast phase was detected as a change in Soret absorbance with a rate constant of 21 000 +/- 4000 s(-)(1) during refolding of cyt c initiated by irradiation of a 308 nm pulse on the NBz-Met65 cyt c in the presence of 2 M GdnHCl. The observed rate constant corresponded well with that reported by the tryptophan fluorescence study [Shastry, M. C. R. S., and Roder, H. (1998) Nat. Struct. Biol. 5, 385-392]. The intermediate decayed with a rate constant of 90 +/- 15, followed by another phase with a rate constant of 13 +/- 3 s(-)(1), and was not seen in the absence of GdnHCl.     

Conversion of the Escherichia coli cytochrome b562 to an archetype cytochrome b: a mutant with bis-histidine ligation of heme iron

A mutant of the Escherichia coli cytochrome b(562) has been created in which the heme-ligating methionine (Met) at position 7 has been replaced with a histidine (His) (M7H). This protein is a double mutant that also has the His 63 to asparagine (H63N) mutation, which removes a solvent-exposed His. While the H63N mutation has no measurable effect on the cytochrome, the M7H mutation converts the atypical His/Met heme ligation in cytochrome b(562) to the classic cytochrome b-type bis-His ligation. This mutation has little effect on the K(d) of heme binding but significantly reduces the chemical and thermal stability of the mutant cytochrome relative to the wild type (wt). Both proteins have similar absorbance (Abs) and electron paramagnetic resonance (EPR) properties characteristic of 6-coordinate low-spin heme. The Abs spectra of the oxidized and reduced bis-His cytochrome are slightly blue-shifted relative to the wt, and the alpha Abs band of ferrous M7H mutant is unusually split. The M7H mutation decreases the midpoint potential of the bound heme by 260 mV at pH 7 and considerably alters the pH dependence of the E(m), which becomes dominated by a single pK(red) = 6.8.     

Structural and kinetic studies of imidazole binding to two members of the cytochrome <Emphasis Type="Italic">c</Emphasis><Subscript>6</Subscript> family reveal an important role for a conserved heme pocket residue

The amino acid at position 51 in the cytochrome c ₆ family is responsible for modulating over 100 mV of heme midpoint redox potential. As part of the present work, the X-ray structure of the imidazole adduct of the photosynthetic cytochrome c ₆ Q51V variant from Phormidium laminosum has been determined. The structure reveals the axial Met ligand is dissociated from the heme iron but remains inside the heme pocket and the Ω-loop housing the Met ligand is stabilized through polar interactions with the imidazole and heme propionate-6. The latter is possible owing to a 180° rotation of both heme propionates upon imidazole binding. From equilibrium and kinetic studies, a Val residue at position 51 increases the stability of the Fe–S(Met) interaction and also affects the dynamics associated with imidazole binding. In this respect, the k _obs for imidazole binding to Arabidopsis thaliana cytochrome c _6A, which has a Val at the position equivalent to position 51 in photosynthetic cytochrome c ₆, was found to be independent of imidazole concentration, indicating that the binding process is limited by the Met dissociation rate constant (about 1 s⁻¹). For the cytochrome c ₆ Q51V variant, imidazole binding was suppressed in comparison with the wild-type protein and the V52Q variant of cytochrome c _6A was found to bind imidazole readily. We conclude that the residue type at position 51/52 in the cytochrome c ₆ family is additionally responsible for tuning the stability of the heme iron–Met bond and the dynamic properties of the ferric protein fold associated with endogenous ligand binding.     

Charge distribution in electron transport components: cytochrome c and cytochrome c oxidase mixtures

Mixtures of cytochrome $\text{c}$ oxidase and cytochrome $\text{c}$ have been titrated by coulometrically generated reductant, methyl viologen radical cation, and physiological oxidant, O₂. Charge distribution among the heme components in mixtures of these two redox enzymes has been evaluated by monitoring the absorbance changes at 605 and 550 nm. Differences in the pathway of the electron transfer process during a reduction cycle as compared to an oxidation cycle are indicated by variations found in the absorbance behavior of the heme components during successive reductive and oxidative titrations. It is apparent that the potential of the cytochrome $\text{a}$ heme is dependent upon whether oxidation or reduction is occurring.     

Spectroscopic identification of the catalytic intermediates of cytochrome c oxidase in respiring heart mitochondria

The catalytic cycle of cytochrome c oxidase (COX) couples the reduction of oxygen to the translocation of protons across the inner mitochondrial membrane and involves several intermediate states of the heme a₃-Cu_B binuclear center with distinct absorbance properties. The absorbance maximum close to 605 nm observed during respiration is commonly assigned to the fully reduced species of hemes a or a₃ (R). However, by analyzing the absorbance of isolated enzyme and mitochondria in the Soret (420–450 nm), alpha (560–630 nm) and red (630–700 nm) spectral regions, we demonstrate that the Peroxy (P) and Ferryl (F) intermediates of the binuclear center are observed during respiration, while the R form is only detectable under nearly anoxic conditions in which electrons also accumulate in the higher extinction coefficient low spin a heme. This implies that a large fraction of COX (>50 %) is active, in contrast with assumptions that assign spectral changes only to R and/or reduced heme a. The concentration dependence of the COX chromophores and reduced c-type cytochromes on the transmembrane potential (ΔΨ_m) was determined in isolated mitochondria during substrate or apyrase titration to hydrolyze ATP. The cytochrome c-type redox levels indicated that soluble cytochrome c is out of equilibrium with respect to both Complex III and COX. Thermodynamic analyses confirmed that reactions involving the chromophores we assign as the P and F species of COX are ΔΨ_m-dependent, out of equilibrium, and therefore much slower than the ΔΨ_m-insensitive oxidation of the R intermediate, which is undetectable due to rapid oxygen binding.