Kinetics of intramolecular electron transfer in cytochrome bo3 from Escherichia coli

We have examined the temperature dependence of the intramolecular electron transfer (ET) between heme b and heme o(3) in CO-mixed valence cytochrome bo(3) (Cbo) from Escherichia coli. Upon photolysis of CO-mixed valence Cbo rapid ET occurs between heme o(3) and heme b with a rate constant of 2.2 x 10(5) s(-1) at room temperature. The corresponding rate of CO recombination is found to be 86 s(-1). From Eyring plots the activation energies for these two processes are found to be 3.4 kcal/mol and 6.7 kcal/mol for the ligand binding and ET reactions, respectively. Using variants of the Marcus equation the reorganization energy (lambda), electronic coupling factor (H(AB)), and the ET distance were found to be 1.4 +/- 0.2 eV, (2 +/- 1) x 10(-3) eV, and 9 +/- 1 A, respectively. These values are quite distinct from the analogous values previously obtained for bovine heart cytochrome c oxidase (CcO) (0.76 eV, 9.9 x 10(-5) eV, 13.2 A). The differences in mechanisms/pathways for heme b/heme o(3) and heme a/heme a(3) ET suggested by the Marcus parameters can be attributed to structural changes at the Cu(B) site upon change in oxidation state as well as differences in electronic coupling pathways between Heme b and heme o(3).     

Direct voltammetric investigation of the electrochemical properties of human hemoglobin: relevance to physiological redox chemistry

Voltammetric measurements on solutions of human hemoglobin using gold electrodes modified with omega-hydroxyalkanethiols have yielded the first direct measure of the reorganization energy of the protein. The value obtained based on extrapolation of the experimentally measured currents, 0.76 eV, is independent of pH (i.e., over the physiologically relevant rage, pH 6.8-7.4) and is remarkably similar to values obtained for myoglobin. This result is perhaps surprising given the marked dependence of the measured reduction potential of hemoglobin on pH (i.e., the redox Bohr effect). Electron transfer rates from the electrode to hemoglobin were also measured. Using similarly measured heterogeneous electron-transfer rates for cytochrome b(5), it is possible to predict the magnitude of the homogeneous electron-transfer rate from cytochrome b(5) to methemoglobin using a formalism developed by Marcus. These predicted rates are in reasonable agreement with reported rates of this physiological reaction based on stopped-flow kinetics experiments. These results suggest that the intrinsic electroreactivity of these heme proteins is sufficient to account for physiologically observed rates. Residual differences between homogeneous phase kinetics and those predicted by heterogeneous phase reactions are suggested to be due to small reductions in the outer-sphere reorganization energy of both component proteins which arise due to solvent exclusion at the interface between the two proteins in complex.     

How does reorganization energy change upon protein unfolding? Monitoring the structural perturbations in the heme cavity of cytochrome c

In several classes of proteins the redox center provides an additional intrinsic biophysical probe that could be used to study the protein structure and function. In present report reorganization energy (lambda, as a parameter describing electron transfer properties) was used to study the protein structural changes around the heme prosthetic group in cytochrome c (cyt c). We attempted to monitor the value of this parameter upon the unfolding process of cyt c by urea, during which it was increased sigmoidally from about 0.52 to 0.82 eV for native and unfold protein, respectively. Results indicate that by structural changes in the heme site, lambda provides a complementary tool for following the unfolding process. Assuming a reversible two-state model for cyt c unfolding, Delta G(H2O), Cm and m values were determined to be 8.32+/-0.7 kcal mol(-1), 1.53+/-0.19 kcalmol(-1)M(-1) and 5.03 M, respectively.     

Effect of decreased ferrochelatase activity on iron and porphyrin content in mitochondria of mice with porphyria induced by griseofulvin

The content of iron and protoporphyrin in liver mitochondria from mice with porphyria induced by griseofulvin was measured. The amount of porphyrin was 0.0076 +/- 0.0043, 4.11 +/- 0.58 and 22.2 +/- 6.8 nmol/mg protein (n = 5) in mitochondria from control animals and animals treated with griseofulvin for 3 days and 4-5 weeks, respectively. The energy coupling of the mitochondria was greatly diminished after 4-5 weeks of treatment, and the ferrochelatase activity was inhibited 80-90%, compared to that of control animals. Mitochondrial preparations isolated by differential centrifugation were contaminated with iron-containing lysosomes which could be removed by Percoll density-gradient centrifugation. In purified mitochondrial preparations no change in the amount of non-heme iron was found after griseofulvin feeding, representing 3.36 +/- 0.15, 3.97 +/- 0.40 and 3.59 +/- 0.23 nmol/mg protein for control animals, 3 days- and 4-5 weeks-treated animals, respectively (n = 4). A mitochondrial iron pool previously identified in rat liver mitochondria and shown to be available for heme synthesis in vitro (Tanger?s, A. (1985) Biochim. Biophys. Acta 843, 199-207) was also present in mitochondria from mice. The magnitude of this iron pool, as well as its availability for heme synthesis, was not changed after treatment of the animals with griseofulvin. The fact that porphyrin, but not iron, accumulated in the mitochondria when ferrochelatase was inhibited is discussed with regard to our understanding of the process of heme synthesis and its regulation.     

Direct electrochemistry of engineered cytochrome b562 molecules with a ligand binding pocket

The rapid and reversible electron transfer reaction of cytochrome b562 was observed at an In2O3 electrode. The estimated heterogeneous electron transfer rate constant (k0') was k0' > or = 5.0 x 10(-3) cm s(-1) at pH 6.5. When the methionine-7 (Met-7) residue, which coordinates to the heme iron as an axial ligand, of the wild-type cytochrome b562 was replaced by an Ala or Gly residue, a water molecule bound to the heme iron and the electron transfer rate constants decreased to 1.3 x 10(-3) and 1.8 x 10(-3) cm s(-1), respectively. This decrease in the electron transfer rate would be due to the larger reorganization energy for the structural change at the redox site. The midpoint potential of cytochrome b562 was shifted negatively by approximately 135 mV by replacing Met-7 with Ala or Gly. Similar dissociation kinetics of cyanide for the mutated molecules as compared to native myoglobin was obtained.     

Factors that determine the unusually low reduction potential of cytochrome c 550 in cyanobacterial photosystem II

A new purification protocol for cytochrome c550 (cyt c550) from His-tagged SYnechocYstis PCC 6803 photosystem II (PSII) was developed which allows the protein to be isolated in high yield and purity. Electron paramagnetic resonance spectroscopy of cyt c550, both free in solution and in intact PSII preparations, yields identical spectra with g values at 1.50, 2.23, and 2.87, which are characteristic for a ferric low-spin bis-histidine coordinated heme. The resonance Raman spectrum of the isolated protein exhibits features characteristic of bis-histidine axial ligation of the iron and a slight ruffling of the heme macrocycle. Together, these results indicate that the heme structure is not very different from most c-type cytochromes, and thus the structure of the heme does not account for its unusually low reduction potential. A direct electrochemical measurement of the reduction potential was performed using square wave voltammetry on a pyrolytic graphite edge electrode, yielding E1.2=-108 mV (vs. NHE) with a peak separation of 5 mV. This value is 150 mV more positive than that previously measured by redox titrations. Because the behavior of the protein in the electrochemistry experiments is indicative of adsorption to the electrode surface, we surmise that binding of the protein to the electrode excludes solvent water from the heme-binding site. We conclude that the degree of solvent exposure makes a significant contribution to the heme reduction potential. Similarly, the binding of cyt c550 to PSII may also reduce the solvent exposure of the heme, and so the direct electrochemical value of the reduction potential may be relevant to the protein in its native state.     

The identification of histidine ligands to cytochrome a in cytochrome c oxidase

A histidine auxotroph of Saccharomyces cerevisiae has been used to metabolically incorporate [1,3-15N2] histidine into yeast cytochrome c oxidase. Electron nuclear double resonance (ENDOR) spectroscopy of cytochrome a in the [15N]histidine-substituted enzyme reveals an ENDOR signal which can be assigned to hyperfine coupling of a histidine 15N with the low-spin heme, thereby unambiguously identifying histidine as an axial ligand to this cytochrome. Comparison of this result with similar ENDOR data obtained on two 15N-substituted bisimidazole model compounds, metmyoglobin-[15N]imidazole and bis[15N]imidazole tetraphenyl porphyrin, provides strong evidence for bisimidazole coordination in cytochrome a.     

Flavin and heme structures in lactate:cytochrome c oxidoreductase: a resonance Raman study

Resonance Raman spectra of Hansenula anomala L-lactate:cytochrome c oxidoreductase (or flavocytochrome b2), of its cytochrome b2 core, and of a bis(imidazole) iron-protoporphyrin complex were obtained at the Soret preresonance from the oxidized and reduced forms. Raman contributions from both the isoalloxazine ring of flavin mononucleotide (FMN) and the heme b2 were observed in the spectra of oxidized flavocytochrome b2. Raman diagrams showing frequency differences of selected FMN modes between aqueous and proteic environments were drawn for various flavoproteins. These diagrams were closely similar for flavocytochrome b2 and for flavodoxins. This showed that the FMN structure must be very similar in both types of proteins, despite their very different proteic pockets. However, the electron density at this macrocycle was found to be higher in flavocytochrome b2 than in these electron transferases. No significant difference was observed between the heme structures in flavocytochrome b2 and in cytochrome b2 core. The porphyrin center-N(pyrrole) distances in the oxidized and reduced heme b2 were estimated to be 1.990 and 2.022 A from frequencies of porphyrin skeletal modes, respectively. The frequency of the vinyl stretching mode of protoporphyrin was found to be very affected in resonance Raman spectra of flavocytochrome b2 and of cytochrome b2 core (1634-1636 cm-1) relative to those observed in the spectra of iron-protoporphyrin [bis(imidazole)] complexes (1620 cm-1). These specificities were interpreted as reflecting a near coplanarity of the vinyl groups of heme b2 with the pyrrole rings to which they are attached. The low-frequency regions of resonance Raman indicated that the iron atoms of the four hemes b2 are in the porphyrin plane whatever their oxidation state. The histidine-Fe-histidine symmetric stretching mode was located at 205 cm-1 in the spectra of flavocytochrome b2 and of cytochrome b2 core. It was insensitive to the iron oxidation state and indicated strong Fe-His bonds in both states.     

Dynamics of electron transfer pathways in cytochrome C oxidase

Cytochrome c oxidase mediates the final step of electron transfer reactions in the respiratory chain, catalyzing the transfer between cytochrome c and the molecular oxygen and concomitantly pumping protons across the inner mitochondrial membrane. We investigate the electron transfer reactions in cytochrome c oxidase, particularly the control of the effective electronic coupling by the nuclear thermal motion. The effective coupling is calculated using the Green's function technique with an extended Huckel level electronic Hamiltonian, combined with all-atom molecular dynamics of the protein in a native (membrane and solvent) environment. The effective coupling between Cu(A) and heme a is found to be dominated by the pathway that starts from His(B204). The coupling between heme a and heme a(3) is dominated by a through-space jump between the two heme rings rather than by covalent pathways. In the both steps, the effective electronic coupling is robust to the thermal nuclear vibrations, thereby providing fast and efficient electron transfer.     

The effect of water on the Fe(3+)/Fe(2+) reduction potential of heme

Hemeproteins can act as catalysts, oxygen carriers or electron conductors. The ferric/ferrous reduction potential E(m7) of iron in the center of the prosthetic group ranges from negative values for peroxidases to an extreme positive value for cytochrome a(3) with Hb and Mb in the middle [1]. Proteins exercise their influence on E(m7) in several ways: via substituents at the periphery of the chelate structure, via the proximal ligand, and via interaction with the surrounding medium, amino acid side chains, or polar solvents. Work on recombined proteins and 2,4-substituted free hemes documented that the first two effects are additive [2]. For the third effect, models of the dielectric media on a molecular level have been successfully applied [3-5]. E(m7) has also been empirically correlated to the degree of heme exposure to water [6-8]. The apoprotein/porphyrin and water/porphyrin interfaces are complementary since water molecules fill any empty space in the crevice and surround any pertinent part of heme outside the protein boundary. The present work links to this idea by a combination of statistical mechanics simulations and quantum mechanical calculations comparing heme in water with heme in an apolar environment. Our results show that polarization of the porphyrin pi-electron cloud by the field from water dipoles influences E(m7). The dominant effect of this and other determinates of iron electron availability is perturbations of delocalized electron density in the porphyrin chelate, reproduced by a model where the prosthetic group is treated as a disc of uniform electron density. The present work is also of interest since the interfacial energy constitutes the main barrier for heme-protein separation [9-11].     

The conformational transition of horse heart porphyrin c

The heme iron of horse heart cytochrome c was selectively removed using anhydrous HF. The product, porphyrin c, exhibits the viscosity, far ultraviolet circular dichroic, and fluorescence properties characteristic for native cytochrome c. However, porphyrin c is more susceptible to denaturation by guanidine hydrochloride and by heat than is the parent cytochrome. All of the conformational parameters of porphyrin c exhibit a common reversible transition centered at 0.95 m guanidine hydrochloride at 23 degrees C and pH 7.0. Guanidine denatured porphyrin c refolds in two kinetic phases having time constants of 20 and 200 ms as detected by stopped flow absorbance or fluorescence measurement, with about 80% of the observed change in the faster phase. The kinetics of porphyrin c refolding are not significantly altered by increasing the viscosity of the refolding solvent 15-fold by addition of sucrose. We suggest that the folding of guanidine denatured cytochrome c is not a diffusion-limited process and that the requirement for protein axial ligation elicits the slow (s) kinetic phase observed in the refolding of cytochrome c.     

Role of heme in structural organization of cytochrome c probed by semisynthesis

The heme prosthetic group of cytochrome c is covalently attached to the protein through thioether bonds to two cysteine side chains. The role of covalent heme attachment to cytochrome c is not understood, and most heme proteins bind the prosthetic group by iron ion ligation and tertiary interactions only. A two-armed attachment seems redundant if the role of covalent connection is to limit heme group orientation or to decouple heme affinity from redox potential. These considerations suggested that one role for covalent attachment of the rigid planar heme might be in organizing the cytochrome c protein structure. Indeed, porphyrin cytochrome c (in which the heme iron ion has been removed) is substantially more ordered than apocytochrome c, having characteristics consistent with a molten globule state. To assess the importance of planar rigidity in ordering this protein, semisynthesis was used to substitute porphyrin by two hydrophobic surrogates, one based on biphenyl and the other on phenanthrene, which have different degrees of planarity and rigidity. The expected two-armed covalent attachment of each surrogate was confirmed in the protein products by a variety of methods including mass spectrometry and NMR. Despite being only about half the size of the porphyrin macrocycle, and lacking any possibility for ligation or polar group interactions with the surrounding protein, the two surrogates confer helix contents that are comparable to that of the molten globule formed by porphyrin cytochrome c under similar solution conditions. The pH titrations of the derivatives monitored by circular dichroism exhibit reversible, bell-shaped folding and unfolding transitions, implying that charge group interactions in the protein are involved in stabilizing the helical structures formed. The thermal transitions of the two derivatives at neutral pH are cooperative, with similar midpoints. The similarity of helical content and structural stability in the two derivatives indicates that the increase in conformational freedom by the biphenyl surrogate does not substantially reduce protein structural stability. The similarity of the two derivatives to porphyrin cytochrome c suggests that the common feature among the three covalently attached groups-their hydrophobicity-is by far the dominant factor in organizing stable structures in the protein.     

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.     

Bile pigment--protein interactions. Coupled oxidation of cytochrome c

A new methodology is described for the chemical modification of the heme prosthetic group of horse heart cytochrome c. The selective modification of the heme moiety of cytochrome c is facilitated by utilizing coupling oxidation conditions. Comparison of the absorption spectra of this chemically modified cytochrome c species in two different solvents (aqueous pyridine and carbon monoxide saturated 6 M guanidinium chloride) with those of two model compounds [bis(pyridine)(2,3,7,8,12,13,17,18-octaethyl-5-oxaporphyrinato)iron(II) tetrafluoroborate salt and (pyridine)carbonyl-(2,3,7,8,12,13,17,18-octaethyl-5-oxaporphyrinato)iron(II) tetrafluoroborate salt] shows that coupled oxidation of cytochrome c affords a new protein with a covalently bound iron(II) oxaporphyrin prosthetic group. Amino acid analysis of this protein-bound iron(II) oxaporphyrin species reveals that only limited modification of the primary structure of the apoprotein occurs during coupled oxidation of cytochrome c. This protein-bound iron(II) oxaporphyrin species is also interconvertible to a protein-bound bilatriene species under hydrolytic conditions. The synthetic utility of the coupled oxidation of cytochrome c for the preparation of chromoproteins which possess covalently bound iron(II) oxaporphyrin and bilatriene prosthetic groups is considered.     

Resonance Raman investigations of chloroperoxidase, horseradish peroxidase, and cytochrome c using Soret band laser excitation.

Resonance Raman spectra of the heme protein chloroperoxidase in its native and reduced forms and complexed with various small ions are obtained by using laser excitation in the Soret region (350-450 nm). Additionally, Raman spectra of horseradish peroxidase, cytochrome P-450cam, and cytochrome c, taken with Soret excitation, are presented and discussed. The data support previous findings that indicate a strong analogy between the active site environments of chloroperoxidase and cytochrome P-450cam. The Raman spectra of native chloroperoxidase are found to be sensitive to temperature and imply that a high leads to low spin transition of the heme iron atom takes place as the temperature is lowered. Unusual peak positions are also found for native and reduced chloroperoxidase and indicate a weakening of porphyrin ring bond strengths due to the presence of a strongly electron-donating axial ligand. Enormous selective enhancements of vibrational modes at 1360 and 674 cm-1 are also observed in some low-spin ferrous forms of the enzyme. These vibrational frequencies are assigned to primary normal modes of expansion of the prophyrin macrocycle upon electronic excitation.     

Fluorescent energy transfer measurements on fluorescein isothiocyanate modified cytochrome P-450 LM2

The distance between the heme iron and the N-terminus of cytochrome P-450 LM2 was determined by fluorescence energy transfer measurements. Fluorescein isothiocyanate which was covalently bound to the N-terminal methionine was used as donor chromophor. The Ro value between fluorescein isothiocyanate and the heme was calculated to be 3.98 nm. The distance between the nitrogen of the N-terminal methionine and the heme was estimated with 2.84 +/- 0.23 nm excluding most likely the N-terminal amino acid of cytochrome P-450 LM2 to participate in the electron transfer to the heme iron. A cytochrome P-450 LM2 membrane model is proposed.     

Conservation of the free energy change of the alkaline isomerization in mitochondrial and bacterial cytochromes c

The thermodynamic parameters of the alkaline transition for oxidized native yeast iso-1 cytochrome c and Rhodopseudomonas palustris cytochrome c(2) (cytc(2)) have been determined through direct electrochemistry experiments carried out at variable pH and temperature and compared to those for horse and beef heart cytochromes c. We have found that both transition enthalpy and entropy are remarkably species dependent, following the order R. palustris cytc(2) > beef (horse) heart cytc>yeast iso-1 cytc. Considering the high homology at the heme-protein interface in the native species, this variability is likely to be mainly determined by differences in the structural and solvation properties and the relative abundance of the various alkaline conformers. Notably, changes in transition enthalpy and entropy among these cytochromes c are compensative and result in small variations in the free energy change of the process (which amounts approximately to +50 kJ mol(-1)) and consequently in the apparent pK(a) value. This compensation indicates that solvent reorganization effects play an important role in the thermodynamics of the transition. This mechanism is functional to ensure a relatively high pK(a) value for the alkaline transition, which is needed to preserve His,Met ligation to the heme iron in cytochrome c at physiological pH and temperature, hence the E(o) value required for the biological function.     

Site-directed mutagenesis of proline 94 to alanine in amicyanin converts a true electron transfer reaction into one that is kinetically coupled

Amicyanin is a type I copper protein that mediates electron transfer (ET) from methylamine dehydrogenase (MADH) to cytochrome c-551i. Pro(94) resides in the "ligand loop" of amicyanin, a sequence of amino acids that contains three of the four copper ligands. ET from the reduced O-quinol tryptophan tryptophylquinone of MADH to oxidized P94A amicyanin is a true ET reaction that exhibits values of electronic coupling (H(AB)) and reorganization energy (lambda) that are the same as for the reaction of native amicyanin. In contrast, the parameters for the ET reaction from reduced P94A amicyanin to oxidized cytochrome c-551i have been significantly altered as a consequence of the mutation. These values of H(AB) and lambda are 8.3 cm(-)(1) and 2.3 eV, respectively, compared to values of 0.3 cm(-)(1) and 1.2 eV for the reaction of native reduced amicyanin. The crystal structure of reduced P94A amicyanin exhibits two alternate conformations with the positions of the copper 1.4 A apart [Carrell, C. J., Sun, D., Jiang, S., Davidson, V. L., and Mathews, F. S. (2004) Biochemistry 43, 9372-9380]. In one of these, conformation B, a water molecule has replaced Met(98) as a copper ligand, and the ET distance to the heme of the cytochrome is increased by 1.4 A. Analysis of these structures suggests that the true k(ET) for ET from the copper in conformation B to heme would be much less than for ET from conformation A. A novel kinetic mechanism is proposed to explain these data in which the reduction of Cu(2+) by methylamine dehydrogenase is a true ET reaction while the oxidation of Cu(1+) by cytochrome c-551i is kinetically coupled ET. By comparison of the temperature dependence of the observed rate of the coupled ET reaction from reduced P94A amicyanin to cytochrome c-551i with the predicted rates and temperature dependence for the true ET reaction from conformation A, it was possible to determine the K(eq) and values of DeltaH degrees and DeltaS degrees that are associated with the non-ET reaction that modulates the observed ET rate.     

Effect of mutation at valine 61 on the three-dimensional structure, stability, and redox potential of cytochrome b5.

To elucidate the role played by Val61 of cytochrome b(5), this residue of the tryptic fragment of bovine liver cytochrome b(5) was chosen for replacement with tyrosine (Val61Tyr), histidine (Val61His), glutamic acid (Val61Glu), and lysine (Val61Lys) by means of site-directed mutagenesis. The mutants Val61Tyr, Val61Glu, Val61His, and Val61Lys exhibit electronic spectra identical to that of the wild type, suggesting that mutation at Val61 did not affect the overall protein structure significantly. The redox potentials determined by differential pulse voltammetry were -10 (wild type), -25 (Val61Glu), -33 (Val61Tyr), 12 (Val61His), and 17 mV (Val61Lys) versus NHE. The thermal stabilities and urea-mediated denaturation of wild-type cytochrome b(5) and its mutants were in the following order: wild type > Val61Glu > Val61Tyr > Val61His > Val61Lys. The kinetics of denaturation of cytochrome b(5) by urea was also analyzed. The first-order rate constants of heme transfer between cytochrome b(5) and apomyoglobin at 20 +/- 0.2 degrees C were 0.25 +/- 0.01 (wild type), 0.42 +/- 0.02 (Val61Tyr), 0.93 +/- 0.04 (Val61Glu), 2.88 +/- 0.01 (Val61His), and 3.88 +/- 0.02 h(-)(1) (Val61Lys). The crystal structure of Val61His was determined using the molecular replacement method and refined at 2.1 A resolution, showing that the imidazole side chain of His61 points away from the heme-binding pocket and extends into the solvent, the coordination distances from Fe to NE2 atoms of two axial ligands are approximately 0.6 A longer than the reported value, and the hydrogen bond network involving Val61, the heme propionates, and three water molecules no longer exists. We conclude that the conserved residue Val61 is located at one of the key positions, the "electrostatic potential" around the heme-exposed area and the hydrophobicity of the heme pocket are determinant factors modulating the redox potential of cytochrome b(5), and the hydrogen bond network around the exposed heme edge is also an important factor affecting the heme stability.     

Cytochrome c interaction with membranes. Absorption and emission spectra and binding characteristics of iron-free cytochrome c.

A cytochrome c derivative from which iron is removed has been prepared and characterized. Several lines of evidence indicate that native and porphyrin cytochrome c have similar conformations: they have similar elution characteristics on Sephadex gel chromatography; in both proteins the tryptophan fluorescence is quenched and the pK values of protonation of the porphyrin are identical. Porphyrin cytochrome c does not substitute for native cytochrome c in either the oxidase reaction or in restoring electron transport in cytochrome-c-depleted mitochondria. It does however competitively inhibit native cytochrome c in these reactions, the Ki for inhibition being larger than the Km for reaction. The absorption and emission spectra, and the polarized excitation spectrum of the porphyrin cytochrome c are characteristic of free base porphyrin. The absence of fluorescence quenching of porphyrin cytochrome c when the protein is bound to cytochrome oxidase suggests that heme to heme distance between these proteins is larger than 0.5 to 0.9 nm depending upon orientation. Binding of the porphyrin cytochrome c to phospholipids or to mitochondria increases the fluorescence polarization of a positively polarized absorption band, which indicates that the bound form of the protein does not rotate freely within the time scale of relaxation from the excited state.