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.     

Effects of charged amino-acid mutation on the solution structure of cytochrome b5 and binding between cytochrome b5 and cytochrome c

The solution structure of oxidized bovine microsomal cytochrome b(5) mutant (E48, E56/A, D60/A) has been determined through 1524 meaningful nuclear Overhauser effect constraints together with 190 pseudocontact shift constraints. The final family of 35 conformers has rmsd values with respect to the mean structure of 0.045+/-0.009 nm and 0.088+/-0.011 nm for backbone and heavy atoms, respectively. A characteristic of this mutant is that of having no significant changes in the whole folding and secondary structure compared with the X-ray and solution structures of wild-type cytochrome b(5). The binding of different surface mutants of cytochrome b(5) with cytochrome c shows that electrostatic interactions play an important role in maintaining the stability and specificity of the protein complex formed. The differences in association constants demonstrate the electrostatic contributions of cytochrome b(5) surface negatively charged residues, which were suggested to be involved in complex formation in the Northrup and Salemme models, have cumulative effect on the stability of cyt c-cyt b(5) complex, and the contribution of Glu48 is a little higher than that of Glu44. Moreover, our result suggests that the docking geometry proposed by Northrup, which is involved in the participation of Glu48, Glu56, Asp60, and heme propionate of cytochrome b(5), do occur in the association between cytochrome b(5) and cytochrome c.     

Cytochrome b5-mediated redox cycling of estrogen

Previously, we have demonstrated microsomal cytochrome P450-catalyzed redox cycling of estrogens. In this study, we investigated the role of cytochrome b5 in redox cycling in order to obtain a full understanding of enzymatic contributions to redox reactions of estrogens. Pure cytochrome P450c and hydrogen peroxide or cumene hydroperoxide oxidized diethylstilbestrol (DES) to diethylstilbestrol-4',4"-quinone (DES Q). This oxidation by H2O2 was doubled by addition of cytochrome b5 to cytochrome P450c (molar ratio of 1:4), but did not proceed with cytochrome b5 alone. The stimulation by cytochrome b5 of the cytochrome P450c-catalyzed oxidation of DES to DES Q occurred via modulation of the Vmax of cytochrome P450c rather than of the Km. DES Q was reduced to DES by purified cytochrome b5 and NADH-dependent cytochrome b5 reductase. Pretreatment of microsomes with an antibody to cytochrome b5 reductase inhibited microsomal NADH-dependent reduction of DES Q to DES by 55%. Cytochrome b5 likely participates in the oxidation of DES to DES Q by interacting with cytochrome P450c and in the reduction of DES Q to DES by interacting with cytochrome b5 reductase. Thus, the study demonstrates that cytochrome b5 plays an active role in biological oxidation and reduction reactions.     

15N chemical shift changes in cytochrome b5: redox-dependent vs. guanidinium chloride-induced changes

The origin of the recently reported chemical shift changes of backbone amide nitrogens of redox proteins upon redox state changes has been investigated. These effects are particularly marked in cytochromes and are clearly present after correction for pseudocontact shifts in the oxidized form (Boyd J, Dobson CM, Morar AS, Williams RJP, Pielak GJ (1999) J Am Chem Soc 121:9247-9248; Guiles RD, Basus VJ, Sarma S, Malpure S, Fox KM, Kuntz ID, Waskell L (1993) Biochemistry 32:8329-8340). 15N-HSQC experiments have been performed on both oxidized and reduced forms of cytochrome b5 in the absence and in the presence of 2 M guanidinium chloride (GdmCl). GdmCl in this concentration is known to sizably alter the structure of the oxidized form of the protein and, in particular, to perturb the hydrogen bonding network. However, the perturbation of the 15N-NMR chemical shift changes is minor compared to the changes occurring upon reduction. It is concluded that changes in hydrogen bonding upon reduction must be modest and cannot account for the observed chemical shift effects. Alternative explanations should thus be looked for.     

Paramagnetism-Based Restraints for Xplor-NIH

Modules that use paramagnetism-based NMR restraints have been developed and integrated in the well known program for solution structure determination Xplor-NIH; the complete set of such modules is called PARArestraints for Xplor-NIH. Paramagnetism-based restraints are paramagnetic relaxation enhancements, pseudocontact shifts, residual dipolar couplings due to metal and overall magnetic anisotropy, and cross correlation between Curie relaxation and nuclear-nuclear dipolar relaxation. The complete program has been tested by back-calculating NOEs and paramagnetism-based restraints from the X-ray structure of cytochrome c (553) from B. pasteurii. Furthermore, the same experimental restraints previously used to determine the solution structure of cytochrome c (553) itself, of cytochrome b (5), and of calbindin D(9k) with the program PARAMAGNETIC DYANA, have been used for structure calculations by using PARArestraints for Xplor-NIH. The agreement between the two programs is quite satisfactory and validates both protocols.     

Solution structure of oxidized microsomal rabbit cytochrome b5. Factors determining the heterogeneous binding of the heme.

Cytochrome b5 is heterogeneous in solution because of the presence of two isomers (A and B), differing in the rotation of the heme plane around the axis defined by the alpha and gamma meso protons. For rabbit cytochrome b5, the A/B ratio is 5 : 1. The solution structure of the major form of the oxidized soluble fragment of rabbit microsomal cytochrome b5 (94 amino acids) is here solved through NMR spectroscopy. From 1908 NOEs, of which 1469 were meaningful, there were 246 pseudocontact shifts and 18 3J couplings, a family of 40 energy-minimized conformers were obtained with average backbone rmsd (for residues 4-84) of 0.060 +/- 0.016 nm and average target function of 0.0078 nm2, no distance violations being larger than 0.03 nm. The structure was compared with the solution structures of the A (major) and B (minor) isomers of the rat cytochrome in the oxidized form. The A/B ratio for the rat cytochrome is 1.5 : 1, despite the very high sequence similarity (93%) to the rabbit protein. This comparison has provided insights into the factors determining the distribution in solution of the two isomers differing with respect to heme orientation. It appears that residues 23 and 74 are both important in determining this distribution, through interaction of their side chains with the prosthetic group. Hydrophobic and steric interactions are the key factors in determining the relative stability of one isomer with respect to the other.     

Solution structure of cytochrome b(5) mutant (E44/48/56A/D60A) and its interaction with cytochrome c.

Using 1617 meaningful NOEs with 188 pseudocontact shifts, a family of 35 conformers of oxidized bovine microsomal cytochrome b5 mutant (E44/48/56A/D60A) has been obtained and is characterized by good resolution (rmsd to the mean structure are 0.047 +/- 0.007 nm and 0.095 +/- 0.008 nm for backbone and heavy atoms, respectively). The solution structure of the mutant, when compared with the X-ray structure of wild-type cytochrome b(5), has no significant changes in the whole folding and secondary structure. The binding between cytochrome b(5) and cytochrome c shows that the association constant of the mutant-cytochrome c complex is much lower than the one for wild-type complex (2.2 x 10(4) M(-1) vs. 5.1 x 10(3) M(-1)). The result suggests the four acidic residues have substantial effects on the formation of the complex between cytochrome b(5) and cytochrome c, and therefore it is concluded reasonably that the electrostatic interaction plays an important role in maintaining the stability and specificity of the complex formed. The competition between the ferricytochrome b(5) mutant and [Cr(oxalate)(3)](3-) for ferricytochrome c shows that site III of cytochrome c, which is a strong binding site to wild-type cytochrome b(5), still binds to the mutant with relatively weaker strength. Our results indicate that certain bonding geometries do occur in the interaction between the present mutant and cytochrome c and these geometries, which should be quite different from the ones of the Salemme and Northrup models.     

Interaction of yeast iso-1-cytochrome c with cytochrome c peroxidase investigated by [15N, 1H] heteronuclear NMR spectroscopy

The interaction of yeast iso-1-cytochrome c with its physiological redox partner cytochrome c peroxidase has been investigated using heteronuclear NMR techniques. Chemical shift perturbations for both 15N and 1H nuclei arising from the interaction of isotopically enriched 15N cytochrome c with cytochrome c peroxidase have been observed. For the diamagnetic, ferrous cytochrome c, 34 amides are affected by binding, corresponding to residues at the front face of the protein and in agreement with the interface observed in the 1:1 crystal structure of the complex. In contrast, for the paramagnetic, ferric protein, 56 amides are affected, corresponding to residues both at the front and toward the rear of the protein. In addition, the chemical shift perturbations were larger for the ferric protein. Using experimentally observed pseudocontact shifts the magnetic susceptibility tensor of yeast iso-1-cytochrome c in both the free and bound forms has been calculated with HN nuclei as inputs. In contrast to an earlier study, the results indicate that there is no change in the geometry of the magnetic axes for cytochrome c upon binding to cytochrome c peroxidase. This leads us to conclude that the additional effects observed for the ferric protein arise either from a difference in binding mode or from the more flexible overall structure causing a transmittance effect upon binding.     

Cytochromeb 50 as a proton carrier in the photosynthetic redox chain of purple bacteria

Recent data on the proton-translocating activity of b cytochromes in chromatophores of purple bacteria and their arrangement in the photosynthetic redox chain are discussed. These data appear to support the concept of the b50 and b-90 cytochrome doublet spanning the membrane. Current schemes of H+ transport by b cytochromes are considered, and the scheme of H+ translocation by cytochrome b50 taking up H+ at the outer side of the membrane and a quinone delivering them from this cytochrome to the inner space of the chromatophore is favored as the most probable in the light of recent findings. This scheme is applicable both to Crofts' linear model of the redox chain and to Mitchell's Q cycle. Kinetic discrepancies between H+ uptake and cytochrome b50 reduction at high ambient redox potentials are interpreted in terms of a special, cytochrome b50-independent, yet Rieske FeS-protein-dependent mode of H+ transport.     

Oxidation of cytochrome b5 by hydroperoxides in rat liver.

1. Spectral changes following the addition of hydroperoxides to isolated hepatocytes and to perfused rat liver were observed. Cytochrome b5 is the major, if not the only, hemoprotein exhibiting redox changes under these conditions; cytochrome b5 is oxidized by added hydroperoxides, e.g. tert-butyl or cumene hydroperoxides. No spectral changes attributable to cytochrome b5 were observed with tert-butanol. 2. The effect is present also when the mitochondrial respiratory chain is inhibited by antimycin A, and it is not observable with isolated mitochondria. On the other hand, the oxidation of cytochrome b5 by hydroperoxides is readily demonstrable in microsomal fractions in presence of NADH. 3. Spectral evidence for a participation of the other microsomal hemoprotein, cytochrome P-450, in the hydroperoxide-linked effects was not obtained. Thus, in hepatocytes from phenobarbital-pretreated rats, no formation of cytochrome P-420, no displacement of a type I substrate, hexobarbital, and no major steady state redox change of cytochrome P-450 was detectable. However, when cytochrome P-450 was dithionite-reduced, an oxidation of this cytochrome occurred upon subsequent hydroperoxide addition. 4. Hydrogen peroxide addition to hepatocytes also leads to a lower steady-state degree of reduction of cytochrome b5. Evidence is provided with hepatocytes from rats pretreated with 3-amino-1,2,4-triazole that H2O2 generated intracellularly, e.g. from added glycolate, also causes a detectable oxidation of cytochrome b5. 5. The mechanism of these hydroperoxide effects remains to be established, and it is not clear whether cytochrome b5 reacts directly or indirectly. However, it is suggested that these effects may be of significance for the further study of cytochrome-b5-linked metabolic pathways.     

Investigation of the solution structures and mobility of oxidised and reduced cytochrome b5 by 2D NMR spectroscopy

Two-dimensional 1H NMR spectroscopy is used to examine the structure and mobility of cytochrome b5 in solution. The assignment of many residues and the interpretation of nuclear Overhauser effects (NOEs) in both redox states allow definition of secondary structural elements. Comparison with X-ray diffraction data shows that differences between crystal and solution structures are small. The dynamics of the protein are examined and the protein is shown to be more mobile than cytochrome c. The relationship of the structure and dynamics to the electron transfer function of cytochrome b5 is discussed.     

Purified cytochrome b561 catalyzes transmembrane electron transfer for dopamine beta-hydroxylase and peptidyl glycine alpha-amidating monooxygenase activities in reconstituted systems

Cytochrome b561 from bovine adrenal medulla chromaffin granules has been purified by fast protein liquid chromatography chromatofocusing. The purified cytochrome was reconstituted into ascorbate-loaded phosphatidylcholine vesicles. With this reconstituted system transmembrane electron transfer for extravesicular soluble dopamine beta-hydroxylase activity was demonstrated. In accordance with the model proposed by Njus et al. (Njus, D., Knoth, J., Cook, C., and Kelley, P. M. (1983) J. Biol. Chem. 258, 27-30), catalytic amounts of a redox mediator were necessary to achieve electron transfer between cytochrome and soluble dopamine beta-hydroxylase. Our observations also showed that when membranous dopamine beta-hydroxylase was reconstituted on cytochrome containing vesicles, electron transfer occurred only in the presence of a redox mediator. Since cytochrome b561 has been found in secretory vesicles associated with peptidyl glycine alpha-amidating monooxygenase, electron transfer to this enzyme was also examined. Analogous to the results obtained for dopamine beta-hydroxylase, transmembrane electron transfer to peptidyl glycine alpha-amidating monooxygenase appears to require a redox mediator between cytochrome and this monooxygenase. These observations indicate that purified cytochrome b561 is capable of providing a transmembrane supply of electrons for both monooxygenases. Since no direct protein to protein electron transfer occurs, the results support the hypothesis that the ascorbate/semidehydroascorbate redox pair serves as a mediator for these enzymes in vivo.     

The thermal stability of the tryptic fragment of bovine microsomal cytochrome b5 and a variant containing six additional residues.

Thermally induced denaturation has been measured for both oxidised and reduced forms of the tryptic fragment of bovine microsomal cytochrome b5 using spectrophotometric methods. In the oxidised state, the tryptic fragment of cytochrome b5 (Ala7-Lys90) denatures in a single cooperative transition with a midpoint temperature (Tm) of approximately 67 degrees C (pH 7.0). The reduced form of the tryptic fragment of cytochrome b5 shows a higher transition temperature of approximately 73 degrees C at pH 7.0 and this is reflected in the values of delta Hm, delta Sm and delta(delta G) of approximately 310kJ.mol-1, 900J.mol-1.K-1 and 5 kJ.mol-1. Increased thermal stability is demonstrated for a variant protein that contains the first 90 amino acid residues of cytochrome b5. These novel increases in stability are observed in both redox states and result from the presence of six additional residues at the amino-terminus. The two forms of cytochrome b5 do not differ significantly in structure with the results suggesting that the reorganisation energy (lambda) of the variant protein, as measured indirectly from redox-linked differences in conformational stability, is small. Consequently the reported subtle differences in reactivity between variants of cytochrome b5 may result from the presence of additional N-terminal residues on the surface of the protein.     

Electron transfer reactions of cytochrome c confined within erythrocyte ghosts

We have prepared and characterized resealed erythrocyte ghosts in which the only discernible pigment is cytochrome c. The resealed ghosts have the normal orientation and are free of 'leaky' species; they are stable and can be maintained at 4 degrees C for many days without lysis. The internal cytochrome c participates in redox reactions with both soluble and insolubilized cytochrome c present externally, and with external cytochrome b5. No reaction was observed with plastocyanin, cytochrome c oxidase or NADPH-cytochrome c reductase. A study has been made of the reaction of the internal cytochrome c with the low molecular weight reductants, ascorbate and glutathione. Complex kinetics are observed with both reagents: with ascorbate the results are best explained by assuming the existence, in the membrane, of a redox-active species able to undergo dedimerization. A protein bound disulfide bond would satisfy the requirement.     

Glycine-231 residue of the mouse mitochondrial protonmotive cytochrome b: mutation to aspartic acid deranges electron transport

The mouse LA9 HQN-R11 cytochrome b mutant, in which the glycine residue at position 231 is replaced by aspartic acid, has increased resistance to all inhibitors of the Qn redox center. It is shown here that this single amino acid alteration has multiple and unexpectedly diverse effects upon the mitochondrial protonmotive bc1 complex. (1) The specific activities of both succinate- and ubiquinol-cytochrome c oxidoreductases in isolated mitochondria are reduced by approximately 65% in the mutant. The parallel reductions in both oxidoreductase activities are not compatible with simple Q pool kinetics for mitochondrial electron transport. (2) There is also a reduction in the relative concentration of cytochrome b in the mutant when calculated on the basis of mitochondrial protein; this decrease does not account for more than a small portion of the reduced catalytic fluxes. (3) The increased antimycin resistance of the mutant is lost upon solubilization by the detergent dodecyl maltoside of the bc1 complex from mitochondria. (4) In pre-steady-state assays of cytochrome b reduction by quinol, the mutant shows a reduced extent of reduction. It was observed in other experiments that there was less oxidant-induced extrareduction of cytochrome b in the mutant. These results could arise from a lowering of the midpoint potentials of both the cytochrome b-562 and cytochrome b-566 heme groups. Alternatively, these effects may reflect changes at the Qp and Qn quinone/quinol binding sites. (5) An unexplained observation for the mutant is the increased rate of cytochrome c1 reduction in the presence of myxothiazol. (6) These functional alterations in the LA9 HQN-R11 mutant are not accompanied by detectable changes in the spectral properties of the cytochrome b or c1 heme groups.     

Amino acid sequence, crystallization and structure determination of reduced and oxidized cytochrome c6 from the green alga Scenedesmus obliquus.

Cytochrome c6from the unicellular green alga Scenedesmus obliquus was sequenced, crystallized in its reduced and oxidized state and the three-dimensional structure of the protein in both redox states was determined by X-ray crystallography. Reduced cytochrome c6crystallized as a monomer in the space group P 21212, whereas the oxidized protein crystallized as a dimer in the space group P 3121. The structures were solved by molecular replacement and refined to 1. 9 and 2.0 A, respectively.Comparison of the structures of both redox states revealed only slight differences on the protein surface, whereas a distortion along the axis between the heme iron and its coordinating Met61 residue was observed. No redox-dependent movement of internal water molecules could be detected. The high degree of similarity of the surfaces and charge distributions of both redox states, as well as the dimerization of cytochrome c6as observed in the oxidized crystal, is discussed with respect to its biological relevance and its implications for the reaction mechanisms between cytochrome c6and its redox partners. The dimer of oxidized cytochrome c6may represent a molecular structure occurring in a binary complex with cytochrome b6f. This assembly might be required for the correct orientation of cytochrome c6with respect to its redox partner cytochrome b6f, facilitating the electron transfer within the complex. If the dimerization is not redox-dependent in vivo, the almost identical surfaces of both redox states do not support a long range differentiation between reduced and oxidized cyt c6, i.e. a random collision model for the formation of an electron transfer complex must be assumed.     

On the oxidation pathways of the mitochondrial bc1 complex from beef heart. Effects of various inhibitors

We have investigated the oxidation of the reduced ubiquinol:cytochrome c reductase (bc1 complex) isolated from beef heart mitochondria. The oxidation of cytochrome c1 by both potassium ferricyanide and cytochrome c in the ascorbate-reduced bc1 complex is not a first-order process. This is taken as evidence that cytochrome c1 is in rapid equilibrium with the Rieske iron-sulphur center. Among the several inhibitors tested, only 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole and stigmatellin are seen to affect this redox equilibrium between the high-potential centers of the beef heart bc1 complex. The oxidation of cytochrome b by cytochrome c in both the succinate-reduced and the fully reduced bc1 complex is blocked by all the inhibitors tested. This inhibition occurs simultaneously with an acceleration in the oxidation of cytochrome c1, even after extraction of the endogenous ubiquinone which is present in the bc1 preparation. Almost complete extraction of ubiquinone from the bc1 complex has no effect upon the rapid phase of cytochrome b oxidation, nor does it alter the inhibition of cytochrome b oxidation by the various inhibitors. The oxidation of cytochrome b by exogenous ubiquinones is stimulated by myxothiazol and partially inhibited by antimycin. However, the addition of both these inhibitors together completely blocks the oxidation of cytochrome b by quinones. In contrast, the simultaneous addition of antimycin and myxothiazol has no such synergistic effect upon the oxidation of cytochrome b by cytochrome c. Our data show that intramolecular electron transfer from cytochrome(s) b to the Rieske iron-sulphur center can take place in the bc1 complex without involvement of endogenous ubiquinone-10. This electron pathway is sensitive to all the inhibitors of the enzyme.     

Stimulation of the respiratory chain of rat liver mitochondria between cytochrome c1 and cytochrome c by glucagon treatment of rats

Mitochondria from glucagon-treated rats oxidize succinate, but not ascorbate plus tetramethylphenylenediamine, faster in the uncoupled state than do control mitochondria. The rate of O(2) uptake in the presence of both substrates is equal to the sum of the rates of the O(2) uptake in the presence of either substrate alone. It is concluded that the mitochondrial respiratory chain is limited at some point between cytochromes b and c and that this step is regulated by glucagon. Measurement of the cytochrome spectra under uncoupled conditions in the presence of succinate and rotenone demonstrates a crossover between cytochromes c and c(1) when control mitochondria are compared with those from glucagon-treated rats, cytochrome c being more oxidized and cytochrome c(1) more reduced in control mitochondria. Under conditions where pyruvate metabolism is studied the control mitochondria are generally more oxidized than those from glucagon-treated rats, the redox state of cytochrome b-566 correlating with the rate of pyruvate metabolism in sucrose medium. However, when the redox state of the mitochondria is taken into account, a crossover between cytochromes c and c(1) is again apparent. The spectra of the b cytochromes are complex, but cytochrome b-562 appears to become more reduced relative to cytochrome b-566 in mitochondria from glucagon-treated rats than in control mitochondria. This can be explained by the existence of a more alkaline matrix in glucagon-treated rats, the redox potential for cytochrome b being pH-sensitive. It is concluded that glucagon stimulates electron flow between cytochromes c(1) and c. The physiological significance of these findings is discussed.     

A comparative structural and functional analysis of cytochrome cM cytochrome c6 and plastocyanin from the cyanobacterium Synechocystis sp. PCC 6803

Cytochrome cM is a new c-class photosynthetic haem protein whose physiological role is still unknown. It has been proposed previously that cytochrome cM can replace cytochrome c6 and plastocyanin in transferring electrons between the two membrane complexes cytochrome b6-f and photosystem I in organisms growing under stress conditions. The experimental evidence herein provided allows us to discard such a hypothesis. We report a procedure to overexpress cytochrome cM from the cyanobacterium Synechocystis sp. PCC 6803 in Escherichia coli cells in mg quantities. This has allowed us to perform a comparative laser flash-induced kinetic analysis of photosystem I reduction by the three metalloproteins from Synechocystis. The bimolecular rate constant for the overall reaction is up to 100 times lower with cytochrome cM than with cytochrome c6 or plastocyanin. In addition, the redox potential value and surface electrostatic potential distribution of cytochrome cM are quite different from those of cytochrome c6 and plastocyanin. These findings strongly indicate that cytochrome cM cannot be recognised by and interact with the same redox partners as the other two metalloproteins.