1H NMR studies of the heme iron coordination in cytochrome c-552 from Euglena gracilis.

The coordination of the heme iron in cytochrome c-552 from Euglena gracilis was investigated by 1H NMR studies at 360 MHz. The data imply that the axial heme ligands are His-14 and Met-56 in both the oxidized and the reduced protein. Studies of mixed solutions of ferro- and ferricytochrome c-552, which provided much of the information on the heme structure, also showed that the intermolecular electron exchange is characterized by a bimolecular rate constant of 5-10(6) mol-1-s-1 at 29 degrees C, which is three orders of magnitude faster than the corresponding reaction in solutions of mammalian cytochromes c.     

MAD structure of Pseudomonas nautica dimeric cytochrome c552 mimicks the c4 Dihemic cytochrome domain association.

The monohemic cytochrome c552from Pseudomonas nautica (c552-Pn) is thought to be the electron donor to cytochrome cd1, the so-called nitrite reductase (NiR). It shows as high levels of activity and affinity for the P. nautica NiR (NiR-Pn), as the Pseudomonas aeruginosa enzyme (NiR-Pa). Since cytochrome c552is by far the most abundant electron carrier in the periplasm, it is probably involved in numerous other reactions. Its sequence is related to that of the c type cytochromes, but resembles that of the dihemic c4cytochromes even more closely.The three-dimensional structure of P. nautica cytochrome c552has been solved to 2.2 A resolution using the multiple wavelength anomalous dispersion (MAD) technique, taking advantage of the presence of the eight Fe heme ions in the asymmetric unit. Density modification procedures involving 4-fold non-crystallographic averaging yielded a model with an R -factor value of 17.8 % (Rfree=20.8 %). Cytochrome c552forms a tight dimer in the crystal, and the dimer interface area amounts to 19% of the total cytochrome surface area. Four tighly packed dimers form the eight molecules of the asymmetric unit.The c552dimer is superimposable on each domain of the monomeric cytochrome c4from Pseudomomas stutzeri (c4-Ps), a dihemic cytochrome, and on the dihemic c domain of flavocytochrome c of Chromatium vinosum (Fcd-Cv). The interacting residues which form the dimer are both similar in character and position, which is also true for the propionates. The dimer observed in the crystal also exists in solution. It has been hypothesised that the dihemic c4-Ps may have evolved via monohemic cytochrome c gene duplication followed by evolutionary divergence and the adjunction of a connecting linker. In this process, our dimeric c552structure might be said to constitute a "living fossile" occurring in the course of evolution between the formation of the dimer and the gene duplication and fusion. The availability of the structure of the cytochrome c552-Pn and that of NiR from P. aeruginosa made it possible to identify putative surface patches at which the docking of c552to NiR-Pn may occur.     

Chromatium vinosum cytochrome c-552. Reduction by photoreduced flavins and intramolecular electron transfer

Cytochrome c-552 from Chromatium vinosum is an unusual heme protein in that it contains two hemes and one flavin per molecule. To investigate whether intramolecular electron transfer occurs in this protein, we have studied its reduction by external photoreduced flavin by using pulsed-laser excitation. This approach allows us to measure reduction kinetics on the mirosecond time scale. Both fully reduced lumiflavin and lumiflavin semiquinone radical reduce cytochrome c-552 with second-order rate constants of approximately 1.4 x 10(6) M-1s-1 and 1.9 x 10(8) M-1 s-1, respectively. Kinetic and spectral data and the results of similar studies with riboflavin indicate that both the flavin and heme moieties of cytochrome c-552 are reduced simultaneously on a millisecond time scale, with the transient formation of a protein-bound flavin anion radical. This is suggested to be due to rapid intramolecular electron transfer. Further, steric restrictions play an important role in the reduction reaction. Studies were conducted on the redox processes following photolysis of CO-ferrocytochrome c-552 in which the flavin was partly oxidized to resolve the kinetics of electron transfer between the heme and flavin of cytochrome c-552. Based on these results, we conclude that intramolecular electron transfer from ferrous heme to oxidized flavin occurs with a first-order rate constant of greater than 1.4 x 10(6) s-1.     

Structural organization of the Chromatium vinosum reaction center associated c-cytochromes.

Magnetic interactions operating between the Chromatium vinosum reaction center associated c-cytochromes and the electron carriers of the reaction center have been assayed by comparing the magnetic properties of these components alone, and in various combinations with paramagnetic forms of the reaction center electron carriers. These studies have yielded the following results. 1. The oxidized paramagnetic forms of the high potential cytochromes c-555 produce no discernable alteration of the light-induced (BChl)2.+signal. 2. Similarly, analysis of the lineshape of the light-induced (BChl)2.+signal shows that a magnetic interaction with the oxidized low potential cytochromes c-553 is likely to produce less than a 1 gauss splitting of the (BChl)2.+signal, which corresponds to a minimum separation of 25 +/- 3 A between the unpaired spins if the heme and (BChl)2 are orientated in a coplanar arrangement, suggesting a minimum separation of 15+/- 3A between the heme edge and the (BChl)2 edge. 3. a prominent magnetic interaction is observed to operate between the cytochrome c-553 and c-555, which results in a 30-35 gauss splitting of these spectra, and suggests an iron to iron separation of about 8 A.4. Magnetic interactions are not observed between the c-cytochromes and the reaction center "primary acceptor" (the iron . quinone complex) nor with the reaction center intermediate electron carrier (which involves bacteriopheophytin) suggesting separations greater than 10 A. 5. Magnetic interactions are not discerned between the two cytochrome c-553 hemes, nor between the two cytochrome c-555 hemes, implying that the distance between the cytochromes of the same pair is greater than 10 A. 6. EPR studies of oriented chromatophores have demonstrated that the cytochrome c-553 and c-555 hemes are perpendicular to each other, and suggest that the cytochrome c-553 heme plane lies parallel to the plane of the membrane, while the cytochrome c-555 heme plane lies perpendicular to the plane of the membrane surface.     

Highly purified hydroxylamine oxidoreductase derived from Nitrosomonas europaea. Some physicochemical and enzymatic properties.

Hydroxylamine oxidoreductase [EC 1.7.3.4] of Nitrosomonas europaea was purified to an electrophoretically homogeneous state and some of its properties were studied. The molecular weight of the enzyme as determined by gel filtration on Sephadex G150 and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate is 175,000-180,000, while the minimum molecular weight per heme determined from the dry weight and heme content is 17,500. The enzyme is a C-type cytochrome; its reduced form shows absorption peaks at 418 (gamma peak), 521 (beta peak), 553 (alpha peak), and 460 nm (due to an unidentified chromophore). Although the alpha peak at 553 nm has a shoulder at 559 nm, the enzyme does not posses protoheme or a cytochrome b subunit. It seems likely that the enzyme molecule possess heme c molecules in different states. The enzyme reacts rapidly with various eukaryotic cytochromes c, but does not react with "bacterial-type" cytochromes c. Although the enzyme does not react with cytochrome c-552 (N. europaea), another C-type cytochrome of the organism, cytochrome c-554 (N. europaea) acts as an electron acceptor for the enzyme.     

Soluble cytochromes from the marine methanotroph Methylomonas sp. strain A4.

Soluble c-type cytochromes are central to metabolism of C1 compounds in methylotrophic bacteria. In order to characterize the role of c-type cytochromes in methane-utilizing bacteria (methanotrophs), we have purified four different cytochromes, cytochromes c-554, c-553, c-552, and c-551, from the marine methanotroph Methylomonas sp. strain A4. The two major species, cytochromes c-554 and c-552, were monoheme cytochromes and accounted for 57 and 26%, respectively, of the soluble c-heme. The approximate molecular masses were 8,500 daltons (Da) (cytochrome c-554) and 14,000 Da (cytochrome c-552), and the isoelectric points were pH 6.4 and 4.7, respectively. Two possible diheme c-type cytochromes were also isolated in lesser amounts from Methylomonas sp. strain A4, cytochromes c-551 and c-553. These were 16,500 and 34,000 Da, respectively, and had isoelectric points at pH 4.75 and 4.8, respectively. Cytochrome c-551 accounted for 9% of the soluble c-heme, and cytochrome c-553 accounted for 8%. All four cytochromes differed in their oxidized versus reduced absorption maxima and their extinction coefficients. In addition, cytochromes c-554, c-552, and c-551 were shown to have different electron paramagnetic spectra and N-terminal amino acid sequences. None of the cytochromes showed significant activity with purified methanol dehydrogenase in vitro, but our data suggested that cytochrome c-552 is probably the in vivo electron acceptor for the methanol dehydrogenase.     

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.     

Structural homology of cytochromes c.

Cytochromes c from many eukaryotic and diverse prokaryotic organisms have been investigated and compared using high-resolution nuclear magnetic resonance spectroscopy. Resonances have been assigned to a large number of specific groups, mostly in the immediate environment of the heme. This information, together with sequence data, has allowed a comparison of the heme environment and protein conformation for these cytochromes. All mitochondrial cytochromes c are found to be very similar to the cytochromes c2 from Rhodospirillaceae. In the smaller bacterial cytochromes, Pseudomonas aeruginosa cytochrome c551 and Euglena gracilis cytochrome c552, the orientation of groups near the heme is very similar, but the folding of the polypeptide chain is different. The heme environment of these two proteins is similar to that of the larger bacterial and mitochondrial cytochromes. Two low-potential cytochromes, Desulfovibrio vulgaris cytochrome c553 and cytochrome c554 from a halotolerant micrococcus have heme environments which are not very similar to those of the other proteins reported here.     

Crystal structure of oxidized Bacillus pasteurii cytochrome c553 at 0.97-A resolution

This article reports the first X-ray structure of the soluble form of a c-type cytochrome isolated from a Gram-positive bacterium. Bacillus pasteurii cytochrome c(553), characterized by a low reduction potential and by a low sequence homology with cytochromes from Gram-negative bacteria or eukaryotes, is a useful case study for understanding the structure-function relationships for this class of electron-transfer proteins. Diffraction data on a single crystal of cytochrome c(553) were obtained using synchrotron radiation at 100 K. The structure was determined at 0.97-A resolution using ab initio phasing and independently at 1.70 A in an MAD experiment. In both experiments, the structure solution exploited the presence of a single Fe atom as anomalous scatterer in the protein. For the 0.97-A data, the phasing was based on a single data set. This is the most precise structure of a heme protein to date. The crystallized cytochrome c(553) contains only 71 of the 92 residues expected from the intact protein sequence, lacking the first 21 amino acids at the N-terminus. This feature is consistent with previous evidence that this tail, responsible for anchoring the protein to the cytoplasm membrane, is easily cleaved off during the purification procedure. The heme prosthetic group in B. pasteurii cytochrome c(553) is surrounded by three alpha-helices in a compact arrangement. The largely exposed c-type heme group features a His-Met axial coordination of the Fe(III) ion. The protein is characterized by a very asymmetric charge distribution, with the exposed heme edge located on a surface patch devoid of net charges. A structural search of a representative set of protein structures reveals that B. pasteurii cytochrome c(553) is most similar to Pseudomonas cytochromes c(551), followed by cytochromes c(6), Desulfovibrio cytochrome c(553), cytochromes c(552) from thermophiles, and cytochromes c from eukaryotes. Notwithstanding a low sequence homology, a structure-based alignment of these cytochromes shows conservation of three helical regions, with different additional secondary structure motifs characterizing each protein. In B. pasteurii cytochrome c(553), these motifs are represented by the shortest interhelix connecting fragments observed for this group of proteins. The possible relationships between heme solvent accessibility and the electrochemical reduction potential are discussed.     

Localization and concentration of hydroxylamine oxidoreductase and cytochromes c-552, c-554, cm-553, cm-552 and a in Nitrosomonas europaea

Two membrane-associated cytochromes, cytochrome c_m-553 and cytochrome c_m-552, were derived from Nitrosomonas europaea. The major c-type cytochrome, cytochrome c_m-553, accounted for 92% of the c heme found in the membrane. It had absorption maxima at 410 nm in the oxidized form and at 417, 523 and 553 nm in the dithionite reduced form. Cytochrome c_m-552 possessed absorption maxima at 409 nm in the oxidized form, at 421, 522 and 552 in the dithionite reduced form, and at 418 in the dithionite reduced plus CO form. The concentration and cellular distribution of the two c-type membrane cytochromes, hydroxylamine oxidoreductase and cytochromes c-552, c-554, and a were determined. Over 95% of the soluble cytochromes (hydroxylamine oxidoreductase cytochromes and c-552 and c-554) were periplasmic, whereas cytochrome c_m-553, cytochrome c_m-552 and cytochrome a were associated with the cell membrane. The outer membrane and cytoplasm were devoid of cytochromes. The extracytoplasmic location of the proton-yielding hydroxylamine oxidizing system (NH₂OH ™ HNO + 2H⁺ + 2e⁻) may contribute to an energy-linked proton gradient. The heme concentrations of hydroxylamine oxidoreductase and cytochromes c-552, c-554, c_m-553, c_m-552 and a were approx. 2.4, 1.2, 0.3, 1.3, 0.1 and 1.1 nmol/mg cell protein, respectively. The corresponding molar ratios of heme were 22:11:2.9:12:1.0:10. The enzyme or cytochrome concentrations for hydroxylamine oxidoreductase and cytochromes c-552, c-554, c_m-553, c_m-552 and a were approx. 0.13, 1.05, 0.09, 0.63, 0.055 and 0.56 nmol/mg cell protein, respectively. The corresponding molar ratios were 0.24:2.2:0.16:1.2:0.1:1.0.     

Electron transfer kinetics between soluble modules of Paracoccus denitrificans cytochrome c1 and its physiological redox partners

The transient electron transfer (ET) interactions between cytochrome c1 of the bc1-complex from Paracoccus denitrificans and its physiological redox partners cytochrome c552 and cytochrome c550 have been characterized functionally by stopped-flow spectroscopy. Two different soluble fragments of cytochrome c1 were generated and used together with a soluble cytochrome c552 module as a model system for interprotein ET reactions. Both c1 fragments lack the membrane anchor; the c1 core fragment (c1CF) consists of only the hydrophilic heme-carrying domain, whereas the c1 acidic fragment (c1AF) additionally contains the acidic domain unique to P. denitrificans. In order to determine the ionic strength dependencies of the ET rate constants, an optimized stopped-flow protocol was developed to overcome problems of spectral overlap, heme autoxidation and the prevalent non-pseudo first order conditions. Cytochrome c1 reveals fast bimolecular rate constants (10(7) to 10(8) M(-1) s(-1)) for the ET reaction with its physiological substrates c552 and c550, thus approaching the limit of a diffusion-controlled process, with 2 to 3 effective charges of opposite sign contributing to these interactions. No direct involvement of the N-terminal acidic c1-domain in electrostatically attracting its substrates could be detected. However, a slight preference for cytochrome c550 over c552 reacting with cyochrome c1 was found and attributed to the different functions of both cytochromes in the respiratory chain of P. denitrificans.     

Structure and heme environment of ferrocytochrome c553 from 1H NMR studies

Cytochrome c553 is a photosynthetic electron transport protein found in algae and cyanobacteria. We have purified cytochromes c553 from five cyanobacteria and studied the structures of the ferrocytochromes by 1H NMR spectroscopy at 360 and 470 MHz. Using standard NMR techniques and by comparing the amino acid sequences of four cytochromes c553 with their 1H NMR spectra, we have assigned in the spectrum of the Aphanizomenon flos-aquae protein 18 resonances to specific amino acid residues and 12 resonances to specific heme protons. Steady state and truncated driven nuclear Overhauser enhancement experiments indicate that a tyrosine and methionine are located near pyrrole ring IV of the heme and that a phenylalanine ring is near the heme alpha-mesoproton. The general folding of the cytochrome c553 protein backbone appears to resemble that of Pseudomonas aeruginosa cytochrome c551, but the chirality of the cytochrome c553 axial methine sulfur is R, the same as that of horse heart cytochrome c.     

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.     

Electron transfer complex between nitrous oxide reductase and cytochrome c552 from Pseudomonas nautica: kinetic, nuclear magnetic resonance, and docking studies

The multicopper enzyme nitrous oxide reductase (N 2OR) catalyzes the final step of denitrification, the two-electron reduction of N 2O to N 2. This enzyme is a functional homodimer containing two different multicopper sites: CuA and CuZ. CuA is a binuclear copper site that transfers electrons to the tetranuclear copper sulfide CuZ, the catalytic site. In this study, Pseudomonas nautica cytochrome c 552 was identified as the physiological electron donor. The kinetic data show differences when physiological and artificial electron donors are compared [cytochrome vs methylviologen (MV)]. In the presence of cytochrome c 552, the reaction rate is dependent on the ET reaction and independent of the N 2O concentration. With MV, electron donation is faster than substrate reduction. From the study of cytochrome c 552 concentration dependence, we estimate the following kinetic parameters: K m c 552 = 50.2 +/- 9.0 muM and V max c 552 = 1.8 +/- 0.6 units/mg. The N 2O concentration dependence indicates a K mN 2 O of 14.0 +/- 2.9 muM using MV as the electron donor. The pH effect on the kinetic parameters is different when MV or cytochrome c 552 is used as the electron donor (p K a = 6.6 or 8.3, respectively). The kinetic study also revealed the hydrophobic nature of the interaction, and direct electron transfer studies showed that CuA is the center that receives electrons from the physiological electron donor. The formation of the electron transfer complex was observed by (1)H NMR protein-protein titrations and was modeled with a molecular docking program (BiGGER). The proposed docked complexes corroborated the ET studies giving a large number of solutions in which cytochrome c 552 is placed near a hydrophobic patch located around the CuA center.     

Comparison of low oxidoreduction potential cytochrome c553 from Desulfovibrio vulgaris with the class I cytochrome c family

The cytochrome c553 from Desulfovibrio vulgaris (DvH c553) is of importance in the understanding of the relationship of structure and function of cytochrome c due to its lack of sequence homology with other cytochromes, and its abnormally low oxido-reduction potential. In evolutionary terms, this protein also represents an important reference point for the understanding of both bacterial and mitochondrial cytochromes c. Using the recently determined nuclear magnetic resonance (NMR) structure of the reduced protein we compare the structural, dynamic, and functional characteristics of DvH c553 with members of both the mitochondrial and bacterial cytochromes c to characterize the protein in the context of the cytochrome c family, and to understand better the control of oxido-reduction potential in electron transfer proteins. Despite the low sequence homology, striking structural similarities between this protein and representatives of both eukaryotic [cytochrome c from tuna (tuna c)] and prokaryotic [Pseudomonas aeruginosa c551 (Psa c551)] cytochromes c have been recognized. The previously observed helical core is also found in the DvH c553. The structural framework and hydrogen bonding network of the DvH c553 is most similar to that of the tuna c, with the exception of an insertion loop of 24 residues closing the heme pocket and protecting the propionates, which is absent in the DvH c553. In contrast, the Psa c551 protects the propionates from the solvent principally by extending the methionine ligand arm. The electrostatic distribution at the recognized encounter surface around the heme in the mitochondrial cytochrome is reproduced in the DvH c553, and corresponding hydrogen bonding networks, particularly in the vicinity of the heme cleft, exist in both molecules. Thus, although the cytochrome DvH c553 exhibits higher primary sequence homology to other bacterial cytochromes c, the structural and physical homology is significantly greater with respect to the mitochondrial cytochrome c. The major structural and functional difference is the absence of solvent protection for the heme, differentiating this cytochrome from both reference cytochromes, which have evolved different mechanisms to cover the propionates. This suggests that the abnormal redox potential of the DvH c553 is linked to the raised accessibility of the heme and supports the theory that redox potential in cytochromes is controlled by heme propionate solvent accessibility.     

Cytochrome c maturation. The in vitro reactions of horse heart apocytochrome c and Paracoccus dentrificans apocytochrome c550 with heme

C-type cytochromes are characterized by having the heme moiety covalently attached via thioether bonds between the heme vinyl groups and the thiols of conserved cysteine residues of the polypeptide chain. Previously, we have shown the in vitro formation of Hydrogenobacter thermophilus cytochrome c(552) (Daltrop, O., Allen, J. W. A., Willis, A. C., and Ferguson, S. J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7872-7876). In this work we report that thioether bonds can form spontaneously in vitro between heme and the apocytochromes c from horse heart and Paracoccus denitrificans via b-type cytochrome intermediates. Both apocytochromes, but not the holo forms, bind 8-anilino-1-naphthalenesulfonate, indicating that the apoproteins each have an affinity for a hydrophobic ligand. Furthermore, for both apocytochromes c an intramolecular disulfide can form between the cysteines of the CXXCH motif that is characteristic of c-type cytochromes. In vitro reaction of these apocytochromes c with heme to yield holocytochromes c, and the tendency to form a disulfide, have implications for the different systems responsible for cytochrome c maturation in vivo in various organisms.     

Comparison of the redox reactions of various types of cytochrome c with iron hexacyanides

The dynamic behavior of various types of cytochromes c in the redox reaction with iron hexacyanides was studied using a temperature-jump method in order to elucidate the molecular mechanism of the redox reaction of cytochromes with their oxidoreductants. Transmittance after the temperature jump changed through a single exponential decay for all cytochromes investigated. Under a constant concentration of anion, the redox reaction of various types of cytochrome c with iron hexacyanides was analyzed according to the scheme: (see formula in text) where C(III) and C(II) are ferric and ferrous cytochromes, respectively, Fe(III) and Fe(II) are ferri- and ferrocyanides, respectively, C(III) . Fe(II) is the ferricytochrome-ferrocyanide complex and C(II) . Fe(III) is the ferrocytochrome-ferricyanide complex. When step B is slower than the other two steps A and C, tau-1 can be represented approximately as (see formula in text) where the bar over the variables denotes the equilibrium value. In a large excess of ferrocyanide against cytochrome, we can estimate kappa 2, kappa-2, K1 and K3 independently. In the case of horse cytochrome c at 18 degrees C in 0.1 M phosphate buffer at pH 7 with 0.3 M KNO3, the estimated parameters are kappa 2 = 100 +/- 50 S-1, kappa-2 = (3.5 +/- 1.0) . 10(3) S-1, K1 = 15 +/- 7 M-1 and K3 = (8.5 +/- 1.5). 10(-4) M. From the same experiments for seven cytochromes (cytochrome c from horse, tuna, Candida krusei, Saccharomyces oviformis, Rhodospirillum rubrum cytochrome c2, Spirulina platensis cytochrome c-554 and Thermus thermophilus cytochrome c-552), the following results can be deduced. (1) Each parameter defined in the scheme above (kappa 2, kappa-2, K1, K3) diverged beyond the error range. Above all, kappa 2 values of cytochromes c-554 and c-552 are as large as 1 . 10(4) S-1 and much larger than those for the other cytochromes (to 50 approx. 700 S-1). (2) The variance of kappa 2K1 and kappa-2/K3 are relatively less than the variances of individual parameters (kappa 2, kappa-2, K1 and K3), which suggests that the values of kappa 2K1 and kappa-2/K3 have been conserved during the course of evolution.     

Laser flash photolysis study of the kinetics of electron transfer reactions of flavocytochrome b2 from Hansenula anomala: further evidence for intramolecular electron transfer mediated by ligand binding.

Intramolecular electron transfer between the heme and flavin cofactors of flavocytochrome b2 is an obligatory step during the enzymatic oxidation of L-lactate and subsequent reduction of cytochrome c. Previous kinetic studies using both steady-state and transient methods have suggested that such intramolecular electron transfer is inhibited when pyruvate, the two-electron oxidation product of L-lactate, is bound at the active site of Hansenula anomala flavocytochrome b2. In contrast to this, we have recently demonstrated using laser flash photolysis that intramolecular electron transfer could be observed in the flavocytochrome b2 from Saccharomyces cerevisiae only when pyruvate was present [Walker, M., & Tollin, G. (1991) Biochemistry 30, 5546-5555], despite a large thermodynamic driving force of 100 mV and apparently favorable cofactor geometry as indicated by crystallographic studies. In the present study, we have utilized laser flash photolysis to investigate intramolecular electron transfer in the flavocytochrome b2 from H. anomala in an effort to address these apparently conflicting interpretations with respect to the influence of pyruvate on enzyme properties. The results obtained are closely comparable to those we reported using the protein from Saccharomyces. Thus, in the absence of pyruvate, bimolecular reduction of both the heme and FMN cofactors by deazaflavin semiquinone occurs (k approximately 10(9) M-1 s-1), followed by a protein concentration dependent intermolecular electron transfer from the semiquinone form of the FMN cofactor to the heme (k approximately 10(7) M-1 s-1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Intramolecular electron transfer from c heme to d1 heme in bacterial cytochrome cd1 nitrite reductase occurs over the same distances at very different rates depending on the source of the enzyme.

Intramolecular electron transfer over 12 A from heme c to heme d(1) was investigated in cytochrome cd(1) nitrite reductase from Pseudomonas aeruginosa, following reduction of the c heme by pulse radiolysis. The rate constant for the transfer is relatively slow, k = 3 s(-1). The present observations contrast with a corresponding rate of electron transfer, 1.4 x 10(3) s(-1), measured for cytochrome cd(1) from Paracoccus pantotrophus, though the relative positions of the two heme groups are the same in both enzymes. The rate of intramolecular electron transfer within the enzyme from P. aeruginosa was accelerated 10(4)-fold (1.4 x 10(4) s(-1)) by the binding of cyanide to the d(1) heme. A coordination change at the d(1) heme upon its reduction is suggested to be a major factor in determining the slow rate of electron transfer in the P. aeruginosa enzyme in the absence of cyanide.     

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.