NMR and EPR studies on a monoheme cytochrome c550 isolated from Bacillus halodenitrificans.

A c-type monoheme ferricytochrome c550 (9.6 kDa) was isolated from cells of Bacillus halodenitrificans sp.nov., grown anaerobically as a denitrifier. The visible absorption spectrum indicates the presence of a band at 695 nm characteristic of heme-methionine coordination. The midpoint redox potential was determined at several pH values by visible spectroscopy. The redox potential at pH 7.6 is 138 mV. When studied by 1H-NMR spectroscopy as a function of pH, the spectrum shows a pH dependence with pKa values of 6.0 and 11.0. According to these pKa values, three forms designated as I, II and III can be attributed to cytochrome c550. The first pKa is probably associated with protonation of the propionate groups. The second pKa value introduces a larger effect in the 1H-NMR spectrum and is probably due to the ionisation of the axial histidine. Studies of temperature variation of the 1H-NMR spectra for both the ferrous and ferri forms of the cytochrome were performed. Heme meso protons, the heme methyl groups, the thioether protons, two protons from a propionate and the methylene protons from the axial methionine were identified in the reduced form. The heme methyl resonances of the ferri form were also assigned. EPR spectroscopy was also used to probe the ferric heme environment. A signal at gmax approximately 3.5 at pH 7.5 was observed indicating an almost axial heme environment. At higher pH values the signal at gmax approximately 3.5 converts mainly to a signal at g approximately 2.96. The pKa associated with this change is around 11.3. The N-terminal sequence of this cytochrome was determined and compared with known amino acid sequences of other cytochromes.     

Thiobacillus ferrooxidans cytochrome c-552: purification and some of its molecular features

Soluble cytochrome c-552 was purified from Thiobacillus ferrooxidans to an electrophoretically homogeneous state. The cytochrome showed absorption peaks at 276, 411 and 523 nm in the oxidized form and peaks at 315, 417, 523 and 552 nm in the reduced form. The molecular weight of the cytochrome was estimated to be 13,800 on the basis of the amino acid composition and heme content, and 14,000 from SDS-polyacrylamide gel electrophoresis analysis. Its midpoint redox potential at pH 7.0 was determined to be +0.36 V. The N-terminal amino acid sequence of the cytochrome was determined as follows: A-G-G-A-G-G-P-A-P-Y-R-I-S-?-D-?-M-V-?-S-G-M-P-G-. Ferrocytochrome c-552 was oxidized by the membrane fraction of T. ferrooxidans, and the oxidation rate was more rapid at pH 3.0 than at pH 6.5. Ferricytochrome c-552 was reduced by Fe(II)-cytochrome c oxidoreductase with Fe2+ at pH 3.5, while horse ferricytochrome c was not reduced by the enzyme under the same reaction conditions.     

Reactivity of photoreduced cytochrome aa3 complexes with molecular oxygen.

Cytochrome c oxidase (ox heart cytochrome aa3) is reduced on illumination in the presence of a photocatalyst system containing deazaflavin and EDTA. The photo-reduced enzyme reacts with oxygen at neutral pH to give a form of ferric enzyme, whereas a corresponding sample partially reduced by light in the absence of any photocatalyst reacts with oxygen to give an oxyferri species ('oxygenated' enzyme). Reduction by the photocatalyst system at an alkaline pH value (9.0) also gives rise to fully reduced oxidase (both haem groups ferrous). At these pH values the immediate product after oxygen addition is a species with a 605-606 nm absorption band, not identical with ferrous cytochrome a, but capable of oxidizing added cytochrome c. This intermediate, which is unstable at neutral pH, may be analogous to the 'compound B' obtained by Chance and co-workers [Chance, Saronio & Leigh (1975) J. Biol. Chem. 250, 9226-9237; Chance, Saronio & Leigh (1979) Biochem. J. 177, 931-941] at low temperatures.     

15N-1H Residual dipolar coupling analysis of native and alkaline-K79A Saccharomyces cerevisiae cytochrome c

Residual dipolar couplings (RDCs) and pseudocontact shifts are experimentally accessible properties in nuclear magnetic resonance that are related to structural parameters and to the magnetic susceptibility anisotropy. We have determined RDCs due to field-induced orientation of oxidized-K79A and reduced cytochrome c at pH 7.0 and oxidized-K79A cytochrome c at pH 11.1 through measurements of amide (15)N-(1)H (1)J couplings at 800 and 500 MHz. The pH 7.0 RDCs for Fe(III)- and Fe(II)-cytochrome c together with available nuclear Overhauser effects were used to recalculate solution structures that were consistent with both sets of constraints. Molecular magnetic susceptibility anisotropy values were calculated for both redox states of the protein. By subtracting the residual dipolar couplings (RDCs) of the reduced form from those of the oxidized form measured at the same magnetic field (800 MHz), we found the RDC contribution of the paramagnetic metal ion in the oxidized protein. The magnetic susceptibility anisotropy, which was calculated from the structure, was found to be the same as that of the paramagnetic metal ion obtained independently from pseudocontact shifts, thereby indicating that the elements of secondary structure either are rigid or display the same mobility in both oxidation states. The residual dipolar coupling values of the alkaline-K79A form are small with respect to those of oxidized native cytochrome, whereas the pseudocontact shifts are essentially of the same magnitude, indicating local mobility. Importantly, this is the first time that mobility has been found through comparison of RDCs with pseudocontact shifts.     

Intergeneric and intrageneric conjugal transfer of plasmids pAMβ1, pIL205 and pIP501 in Lactobacillus sake

Abstract Three membrane-bound acid-stable cytochromes c with molecular masses of 46, 30 and 21 kDa were characterized from a new Thiobacillus ferrooxidans strain. They were solubilized with high concentrations of dodecylmaltoside at pH 8. The 30 kDa cytochrome c was purified to a homogeneous state as established by SDS-PAGE analysis. It showed an absorption peak at 410 nm in the oxidized form and at 418, 523 and 552 nm in the reduced form. The 46 kDa cytochrome c co-purified with a non-heme protein of 36 kDa. The amino acid composition and the N-terminal amino acid sequence of the 46 kDa cytochrome c were determined and compared with those of the soluble 14 kDa and the membrane-bound 21, 22.3 and 68 kDa cytochromes c isolated from two different strains. The results clearly show that this cytochrome is distinct from both the 22.3, 21 and 14 kDa cytochrome species, and exhibits some similarities with the 68 kDa cytochrome c as regards its amino acid composition.     

Effect of membrane potential and pH gradient on electron transfer in cytochrome oxidase

Steady-state spectra of cytochrome oxidase in phospholipid vesicles were obtained by using hexaammineruthenium(II) and ascorbate as reductants. Cytochrome a was up to 80% reduced in the steady state in coupled vesicles. Upon addition of nigericin or acetate, which decrease delta pH, resulting in an increase in delta psi, cytochrome a became more oxidized in the steady state with no change in the rate of respiration. On the other hand, uncouplers or valinomycin plus nigericin, which lower both delta psi and delta pH, stimulated respiration 2-8-fold and also lowered the steady-state level of reduction of cytochrome a. These experiments indicate that electron transfer between cytochromes a and a 3 is sensitive primarily to the pH gradient. Studies with the reconstituted and the soluble enzyme at various pH values indicated that the pH on the matrix side of the membrane, rather than delta pH, controlled the steady-state level of reduced cytochrome a. Hexaammineruthenium(II) substituted for cytochrome c in measurements of proton pumping by cytochrome oxidase. Dicyclohexylcarbodiimide, which eliminated proton pumping by cytochrome oxidase, decreased the effect of ionophores on the steady-state level of reduced cytochrome a.     

Characteristics of the redox-linked proton ejection in beef-heart cytochrome c oxidase reconstituted in liposomes

In this paper a study is presented of the characteristics of redox-linked proton ejection exhibited by isolated beef-heart cytochrome c oxidase incorporated in asolectin vesicles. The enzyme was 90% oriented 'right-side out' as in the mitochondrial membrane. The effects on the H+/e- stoichiometry of the modalities of activation of electron flow, the pH of the medium and its ionic composition were investigated. The results obtained show that, whilst ferrocytochrome c pulses of the aerobic oxidase vesicles at neutral pH and in the presence of saturating concentrations of valinomycin and K+ to ensure charge compensation produced H+/e- ratios around 1 (as has been shown previously), oxygen pulses of reduced anaerobic vesicles supplemented with cytochrome c, gave H+/e- ratios around 0.3. The H+/e- ratios exhibited, with both reductant and oxidant pulses, a marked pH dependence. Maximum values were observed at pH 7.0-7.7, which decreased to negligible values at acidic pH with apparent pKa of 6.7-6.3. Mg2+ and Ca2+ caused a marked depression of the H+/e- ratio, which in the presence of these cations and after a few ferrocytochrome pulses, became negligible. Analysis of cytochrome c oxidation showed that the modalities of activation of electron flow and divalent cations exerted profound effects on the kinetics of cytochrome c oxidation by oxidase vesicles. The observations presented seem to provide interesting clues for the nature and mechanism of redox-linked proton ejection in reconstituted cytochrome c oxidase.     

Purification and characterization of the cytochrome oxidase from alkalophilic Bacillus firmus RAB

A cytochrome oxidase was purified 52-fold from membranes of alkalophilic Bacillus firmus RAB by extraction with Triton X-100, ion-exchange and hydroxyapatite chromatography, and gel filtration. On denaturing gels, the purified enzyme dissociated into two subunits of 56,000 and 40,000 Mr as well as a cytochrome c with an Mr of approximately 14,000. Heme contents calculated for an enzyme with a molecular weight of 110,000 were found to be 2 mol of heme a and 1 mol of heme c per mol of cytochrome oxidase; approximately 2 mol of copper per mol of purified enzyme was also found. Enzyme activity was observed in assays using reduced yeast or horse heart cytochrome c. Activity of the purified enzyme was optimal at pH 6.0 and in the presence of added lipids. Impure, membrane-associated activity exhibited a broader pH range for optimal activity extending to alkaline values.     

Spin-equilibrium and heme-ligand alteration in a high-potential monoheme cytochrome (cytochrome c554) from Achromobacter cycloclastes, a denitrifying organism

A c-type monoheme cytochrome c554 (13 kDa) was isolated from cells of Achromobacter cycloclastes IAM 1013 grown anaerobically as a denitrifier. The visible absorption spectrum indicates the presence of a band at 695 nm characteristic of heme-methionine coordination (low-spin form) coexisting with a minor high-spin form as revealed by the contribution at 630 nm. Magnetic susceptibility measurements support the existence of a small contribution of a high-spin form at all pH values, attaining a minimum at intermediate pH values. The mid-point redox potential determined by visible spectroscopy at pH 7.2 is +150 mV. The pH-dependent spin equilibrum and other relevant structural features were studied by 300-MHz 1H-NMR spectroscopy. In the oxidized form, the 1H-NMR spectrum shows pH dependence with pKa values at 5.0 and 8.9. According to these pKa values, three forms designated as I, II and III can be attributed to cytochrome c554. Forms I and II predominate at low pH values, and the 1H-NMR spectra reveal heme methyl proton resonances between 40 ppm and 22 ppm. These forms have a methionyl residue as a sixth ligand, and C6 methyl group of the bound methionine was identified in the low-field region of the NMR spectra. Above pH 9.6, form III predominates and the 1H-NMR spectrum is characterized by down-field hyperfine-shifted heme methyl proton resonances between 29 ppm and 22 ppm. Two new resonances are observed at congruent to 66 ppm and 54 ppm, and are taken as indicative of a new type of heme coordination (probably a lysine residue). These pH-dependent features of the 1H-NMR spectra are discussed in terms of the heme environment structure. The chemical shifts of the methyl resonances at different pH values exhibit anti-Curie temperature dependence. In the ferrous state, the 1H-NMR spectrum shows a methyl proton resonance at -3.9 ppm characteristic of methionine axial ligation. The electron-transfer rate between ferric and ferrous forms has been estimated to be smaller than 2 x 10(4) M-1 s-1 at pH 5. EPR spectroscopy was also used to probe the ferric heme environment. A prominent signal at gmax congruent to 3.58 and the overall lineshape of the spectrum indicate an almost axial heme environment.     

The heme iron coordination of unfolded ferric and ferrous cytochrome c in neutral and acidic urea solutions. Spectroscopic and electrochemical studies

The heme iron coordination of unfolded ferric and ferrous cytochrome c in the presence of 7-9 M urea at different pH values has been probed by several spectroscopic techniques including magnetic and natural circular dichroism (CD), electrochemistry, UV-visible (UV-vis) absorption and resonance Raman (RR). In 7-9 M urea at neutral pH, ferric cytochrome c is found to be predominantly a low spin bis-His-ligated heme center. In acidic 9 M urea solutions the UV-vis and near-infrared (NIR) magnetic circular dichroism (MCD) measurements have for the first time revealed the formation of a high spin His/H(2)O complex. The pK(a) for the neutral to acidic conversion is 5.2. In 9 M urea, ferrous cytochrome c is shown to retain its native ligation structure at pH 7. Formation of a five-coordinate high spin complex in equilibrium with the native form of ferrous cytochrome c takes place below the pK(a) 4.8. The formal redox potential of the His/H(2)O complex of cytochrome c in 9 M urea at pH 3 was estimated to be -0.13 V, ca. 100 mV more positive than E degrees ' estimated for the bis-His complex of cytochrome c in urea solution at pH 7.     

Formation of an iso-1-cytochrome c-like species containing a covalently bonded heme group from the apoprotein by a yeast cell-free system in the presence of hemin

Incubation of the 125I-labeled apoprotein, prepared from 125I-labeled iso-1-cytochrome c, with a yeast mitochondrial fraction in the presence of hemin, NADPH, and an extract of the postmitochondrial fraction at 32 +/- 1 degree C for 30 min has resulted in formation of cytochrome c-like species in yields of up to 35%. This radioactive synthesized species contains a functional group which responds to reduction with ascorbate and oxidation with K3Fe(CN)6 in that it is resistant in the reduced form and susceptible in the oxidized form to trypsin action in a manner characteristic of native cytochrome c. The functional group cannot be removed from the protein by cold HCl-acetone or 8 M urea treatment. The reduced form of the synthesized species exhibits resistance against autoxidation and the oxidized form can be reduced also by cytochrome b2. The synthesized species exhibits the same compact hydrodynamic volume of native cytochrome c. Treatment with silver sulfate followed by incubation with dithiothreitol converts the synthesized species to the original apoprotein as judged by an increase in the hydrodynamic volume. Thus, the synthesized species is indistinguishable from the original labeled iso-1-cytochrome c by these measurements; i.e. the synthesized species consists of the apoprotein to which heme is covalently attached through the thioether bond(s). The active factor of the mitochondrial fraction is heat-labile. The synthetic activity is strongly dependent on pH with a maximum approximately at pH 7.0. Hemin (or heme) appears to be required for this synthesis. The postmitochondrial fraction is inactive by itself. However, its addition markedly increases the synthetic activity. This factor is heat-stable, soluble in 80% methanol (or 75% ethanol), and insoluble in ethyl ether or ethyl acetate. Addition of NADP(H) (or NAD(H)) also increases the synthetic activity, the reduced form being more effective than the oxidized form. The postmitochondrial factor and the pyridine nucleotides appear to enhance the effect of each other. Thus, it seems that cytochrome c or a cytochrome c-like species is formed from the apoprotein and heme (or hemin) by an enzyme, cytochrome c synthetase, present in mitochondria.     

The kinetics of electron transfer between pseudomonas aeruginosa cytochrome c-551 and its oxidase.

The redox reaction between cytochrome c-551 and its oxidase from the respiratory chain of pseudomonas aeruginosa was studied by rapid-mixing techniques at both pH7 and 9.1. The electron transfer in the direction of cytochrome c-551 reduction, starting with the oxidase in the reduced and CO-bound form, is monophasic, and the governing bimolecular rate constants are 1.3(+/- 0.2) x 10(7) M-1 . s-1 at pH 9.1 and 4 (+/- 1) x 10(6) M-1 . s-1 at pH 7.0. In the opposite direction, i.e. mixing the oxidized oxidase with the reduced cytochrome c-551 in the absence of O2, both a lower absorbance change and a more complex kinetic pattern were observed. With oxidized azurin instead of oxidized cytochrome c-551 the oxidation of the c haem in the CO-bound oxidase is also monophasic, and the second-order rate constant is 2 (+/- 0.7) x 10(6) M-1 . s-1 at pH 9.1. The redox potential of the c haem in the oxidase, as obtained from kinetic titrations of the completely oxidized enzyme with reduced azurin as the variable substrate, is 288 mV at pH 7.0 and 255 mV at pH 9.1. This is in contrast with the very high affinity observed in similar titrations performed with both oxidized azurin and oxidized cytochrome c-551 starting from the CO derivative of the reduced oxidase. It is concluded that: (i) azurin and cytochrome c-551 are not equally efficient in vitro as reducing substrates of the oxidase in the respiratory chain of Pseudomonas aeruginosa; (ii) CO ligation to the d1 haem in the oxidase induces a large decrease (at least 80 mV) in the redox potential of the c-haem moiety.     

Electron transfer between cytochrome a and copper A in cytochrome c oxidase: a perturbed equilibrium study

Intramolecular electron transfer in partially reduced cytochrome c oxidase has been studied by the perturbed equilibrium method. We have prepared a three-electron-reduced, CO-inhibited form of the enzyme in which cytochrome a and copper A are partially reduced and in an intramolecular redox equilibrium. When these samples were irradiated with a nitrogen laser (0.6-ns, 1.0-mJ pulses) to photodissociate the bound CO, changes in absorbance at 598 and 830 nm were observed which were consistent with a fast electron transfer from cytochrome a to copper A. The absorbance changes at 598 nm gave an apparent rate of 17,000 +/- 2000 s-1 (1 sigma), at pH 7.0 and 25.5 degrees C. These changes were not observed in either the CO mixed-valence or the CO-inhibited fully reduced forms of the enzyme. The rate was fastest at about pH 8.0, falling off toward both lower and higher pHs. There was a small but clear temperature dependence. The process was also observed in the cytochrome c-cytochrome c oxidase high-affinity complex. The electron equilibration measured between cytochrome a and copper A is far faster than any rate measured or inferred previously for this process.     

Characterization of electron donation to cytochrome c-555 in Chromatium vinosum from ferrocyanide, tetramethylphenylenediamine and reduced dimethylquinone. Effects of redox potential, pH and salt concentration

1. The dependences of the reduction of ferricytochrome c-555 in the reaction center-cytochrome c complex on the redox potential and pH were investigated using N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), ferrocyanide, and reduced 2,5-dimethyl-p-quinone as electron donors. 2. In the reduction of cytochrome c-555 by TMPD, the unprotonated form was the exclusive electron donor to the cytochrome with a second-order rate constant of 1.0 X 10(5) M-1.s-1. 3. Ferrocyanide reduced cytochrome c-555 slowly with a rate constant of 7.8 X 10(3) M-1.s-1 at infinite salt concentration. The value of -5.2 X 10(-4) elementary charge/A2 was estimated as the surface charge density in the vicinity of cytochrome c-555 by analyzing the salt effect on the cytochrome reduction using the Gouy-Chapman theory. 4. The characteristics of the dependences of the reduction of cytochrome c-555 by reduced 2,5-dimethyl-p-quinone on the redox potential and pH were well explained by the redox potential and pH dependences of the formation of the semiquinone. In the neutral-to-alkaline pH range the anionic semiquinone was the main electron-donating species with a second-order rate constant of 6.0 X 10(7) m-1.s-1.     

A Candida albicans metallopeptidase degrades constitutive proteins of extracellular matrix

A soluble c-type cytochrome was first purified from Geobacter metallireducens to an electrophoretically homogeneous state. The purified cytochrome c showed absorption peaks at 530 and 409 nm in the oxidized form and 552, 522, and 418 nm in the reduced form. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate allowed us to calculate the molecular mass at 9.5 kDa. It contained 3 mol of heme c per molecule of the protein on the basis of heme c and protein concentration. The mid-point redox potential at pH 7.0 was determined to be -190 mV. Although the N-terminal amino acid sequence of the first 17 residues was similar to that of Desulfuromonas acetoxidans cytochrome c7, G. metallireducens cytochrome c did not show Fe(III)-reducing activity.     

Cytochrome c peroxidase activity of a protease-modified form of cytochrome c-552 from the denitrifying bacterium Pseudomonas perfectomarina

Protease activity present in aerobically grown cells of Pseudomonas perfectomarina, protease apparently copurified with cytochrome c-552, and trypsin achieved a limited proteolysis of the diheme cytochrome c-552. That partial lysis conferred cytochrome c peroxidase activity upon cytochrome c-552. The removal of a 4000-Da peptide explains the structural changes in the cytochrome c-552 molecule that resulted in the appearance of both cytochrome c peroxidase activity (with optimum activity at pH 8.6) and a high-spin heme iron. The oxidized form of the modified cytochrome c-552 bound cyanide to the high-spin ferric heme with a rate constant of (2.1 +/- 0.1) X 10(3) M-1 s-1. The dissociation constant was 11.2 microM. Whereas the intact cytochrome c-552 molecule can be half-reduced by ascorbate, the cytochrome c peroxidase was not reducible by ascorbate, NADH, ferrocyanide, or reduced azurin. Dithionite reduced the intact protein completely but only half-reduced the modified form. The apparent second-order rate constant for dithionite reduction was (7.1 +/- 0.1) X 10(2) M-1 s-1 for the intact protein and (2.2 +/- 0.1) X 10(3) M-1 s-1 for the modified form. In contrast with other diheme cytochrome c peroxidases, reduction of the low-spin heme was not necessary to permit ligand binding by the high-spin heme iron.     

Fusion of phospholipid vesicles mediated by cytochrome c

Fusion of phosphatidylserine/phosphatidylethanolamine (1/1) vesicles induced by cytochrome c is studied at a wide range of pH values. A pH profile for the fusion with maximum values at pH 5 and pH 8 is obtained and this is found to be similar to the profile for cytochrome c binding to the vesicles. The binding property of apocytochrome c to the same phospholipid vesicles is found to be about the same as that of the cytochrome c at low ionic strength, but very different at high salt concentrations. No appreciable fusion of vesicles by apocytochrome c is observed. Proteolytic treatment and dansyl chloride labeling of cytochrome c- and apocytochrome c-vesicle complexes show that the C-terminal segments of these proteins with molecular weights of about 3000 and 5000, respectively, penetrate the bilayer. The hydrophobic labeling studies with photoreactive phosphatidylcholine in the bilayer show that segments of both cytochrome c and apocytochrome c go deep into the bilayer.     

Steady state kinetics and binding of eukaryotic cytochromes c with yeast cytochrome c peroxidase.

1. The steady state kinetics for the oxidation of ferrocytochrome c by yeast cytochrome c peroxidase are biphasic under most conditions. The same biphasic kinetics were observed for yeast iso-1, yeast iso-2, horse, tuna, and cicada cytochromes c. On changing ionic strength, buffer anions, and pH, the apparent Km values for the initial phase (Km1) varied relatively little while the corresponding apparent maximal velocities varied over a much larger range. 2. The highest apparent Vmax1 for horse cytochrome c is attained at relatively low pH (congruent to 6.0) and low ionic strength (congruent to 0.05), while maximal activity for the yeast protein is at higher pH (congruent to 7.0) and higher ionic strength (congruent to 0.2), with some variations depending on the nature of the buffering ions. 3. Direct binding studies showed that cytochrome c binds to two sites on the peroxidase, under conditions that give biphasic kinetics. Under those ionic conditions that yield monophasic kinetics, binding occurred at only one site. At the optimal buffer concentrations for both yeast and horse cytochromes c, the KD1 and KD2 values approximate the Km1 and Km2 values. At ionic strengths below optimal, binding becomes too strong and above optimal, too weak. 4. Under ionic conditions that are optimal and give monophasic kinetics with horse cytochrome c but are suboptimal for the yeast protein, yeast cytochrome c strongly inhibits the reaction of horse cytochrome c with peroxidase, uncompetitively at one site and competitively at a second site. The appearance of the second site under monophasic conditions is interpreted as an allosteric effect of the inhibitor binding to the first site. 5. The simplest model accounting for these observations postulates two kinetically active sites on each molecule of peroxidase, a high affinity and a low affinity site, that may correspond to the free radical and the heme iron (IV) of the oxidized enzyme, respectively. Both oxidizing equivalents may be discharged at either site. Furthermore, the enzyme appears to exist as an equilibrium mixture of a high ionic strength form, EH and a low ionic strength form, EL, the former reacting optimally with yeast cytochrome c, and the latter with horse cytochrome c.     

Equilibrium unfolding of a small low-potential cytochrome, cytochrome c553 from Desulfovibrio vulgaris.

To understand general aspects of stability and folding of c-type cytochromes, we have studied the folding characteristics of cytochrome c553 from Desulfovibrio vulgaris (Hildenborough). This cytochrome is structurally similar but lacks sequence homology to other heme proteins; moreover, it has an abnormally low reduction potential. Unfolding of oxidized and reduced cytochrome c553 by guanidine hydrochloride (GuHCl) was monitored by circular dichroism (CD) and Soret absorption; the same unfolding curves were obtained with both methods supporting that cytochrome c553 unfolds by an apparent two-state process. Reduced cytochrome c553 is 7(3) kJ/mol more stable than the oxidized form; accordingly, the reduction potential of unfolded cytochrome c553 is 100(20) mV more negative than that of the folded protein. In contrast to many other unfolded cytochrome c proteins, upon unfolding at pH 7.0 both oxidized and reduced heme in cytochrome c553 become high-spin. The lack of heme misligation in unfolded cytochrome c553 implies that its unfolded structure is less constrained than those of cytochromes c with low-spin, misligated hemes.     

Characterization of the reaction between ferrocytochrome c and cytochrome c oxidase.

The reaction between cytochrome c oxidase and ferrocytochrome c has been investigated by the stopped-flow method. It has been found that only one electron acceptor, a heme group, in the oxidase is rapidly reduced by cytochrome c. The presence of N3- does not affect the reduction of the acceptor, which supports the hypothesis that this is identical with cytochrome a. The results are consistent with the existence of a simple equilibrium between cytochrome a and cytochrome c: c-2 + a-3+ in equilibrium c-3+ + a-2+ with an equilibrium constant corresponding to an oxidation-reduction potential of cytochrome a 30 mV higher than that for cytochrome c at pH 7.4. The oxidation-reduction potential of the a-3+ /a-2+ couple, 285 mV (based on a potential of 255 mV for cytochrome c), and the optical properties of the reduced form indicate that it is identical with neither of the reduced hemes seen in potentiometric titrations. The oxidase species resulting from the rapid reduction of cytochrome a by cytochrome c is proposed to represent a metastable intermediate state which, under anaerobic conditions, eventually is transformed into a more stable state characterized by a reduced high-potential heme.