Gene synthesis, bacterial expression, and 1H NMR spectroscopic studies of the rat outer mitochondrial membrane cytochrome b5.

The gene coding for the water-soluble domain of the outer mitochondrial membrane cytochrome b5 (OM cytochrome b5) from rat liver has been synthetized and expressed in Escherichia coli. The DNA sequence was obtained by back-translating the known amino acid sequence [Lederer, F., Ghrir, R., Guiard, B., Cortial, S., & Ito, A. (1983) Eur. J. Biochem. 132, 95-102]. The recombinant OM cytochrome b5 was characterized by UV-visible, EPR, and 1H NMR spectroscopy. The UV-visible and EPR spectra of the OM cytochrome b5 are almost identical to the ones obtained from the overexpressed rat microsomal cytochrome b5 [Bodman, S. B. V., Schyler, M. A., Jollie, D. R., & Sligar, S. G. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9443-9447]. The one-dimensional 1H NMR spectrum of the OM cytochrome b5 indicates that the rhombic perturbation of the ferric center is essentially identical to that in the microsomal beef, rabbit, chicken, and rat cytochromes b5. Two-dimensional 1H NMR spectroscopy (NOESY) and one-dimensional NOE difference spectroscopy were used to assign the contact-shifted resonances that correspond to each of the two isomers that result from the rotation of the heme around its alpha-gamma-meso axis. The assignment of the resonances allowed the determination of the heme orientation ratio in the OM cytochrome b5, which was found to be 1.0 +/- 0.1. It is noteworthy that the two cytochromes b5 that have similar populations of the two heme isomers (large heme disorder) originate from the rat liver.     

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.     

Solution structure of the B form of oxidized rat microsomal cytochrome b5 and backbone dynamics via 15N rotating-frame NMR-relaxation measurements. Biological implications.

Cytochrome b5 in solution has two isomers (A and B) differing by a 180 degrees rotation of the protoporphyrin IX plane around the axis defined by the alpha and gamma meso protons. Homonuclear and heteronuclear NMR spectroscopy has been employed in order to solve the solution structure of the minor (B) form of the oxidized state of the protein and to probe its backbone dynamics in the microsecond--ms timescale in both oxidation states. A family of 40 conformers has been obtained using 1302 meaningful NOEs and 220 pseudocontact shifts and is characterized by high quality and good resolution (rmsd to the mean structure of 0.055 +/- 0.009 nm and 0.103 +/- 0.011 nm for backbone and heavy atoms, respectively). Extensive comparisons of the structural and dynamics changes associated with the A-to-B form interconversion for both oxidation states were subsequently performed. Propionate 6 experiences a redox-state-dependent reorientation as does propionate 7 in the A form. Significant insights are obtained into the role of the protein frame for efficient biological function and backbone mobility is proposed to be one of the factors that could control the reduction potential of the heme.     

Characterization of cytochrome b5 reconstituted with a ferric chlorin and a ferric oxochlorin

The role of the electronic properties of the heme group of rat cytochrome b5 in biological electron transfer was investigated by substituting chlorin analogues for the native protoporphyrin IX prosthetic group. The resultant purified proteins displayed physical and chemical properties distinct from those of the native enzyme. Optical spectroscopy of the ferric chlorin substituted cytochrome b5 revealed a blue-shifted Soret at 404 nm and a band at 586 nm characteristically red-shifted from the protohemin absorption band. The reduced, reconstituted protein displayed maxima at 406, 418, 563, and 600 nm. The oxidized cytochrome b5 containing the oxochlorin analogue produced a red-shifted Soret with maxima at 338, 416, and 602 nm. The reduced species differed only in the visible region with absorption maxima at 508, 554, and 600 nm. Characterization by EPR spectroscopy of the oxochlorin-substituted cytochrome b5 yielded g values of 2.566, 2.375, and 1.756 and respective axial delta/lambda and rhombic V/lambda components of 2.857 and 3.287, indicating significant electronic distortion in the chlorin ring and an increase in electron donation from the axial histidine ligands. A decrease in the reduction potential of 52 +/- 5 mV (50 mM KPi, pH 7.0, 25 degrees C) for the chlorin-reconstituted cytochrome b5 was determined with respect to that of native cytochrome b5. The reduction potential for the oxochlorin-containing cytochrome b5 was unchanged from that of the native system. Both of the reconstituted proteins were found to be capable of transferring electrons to cytochrome c in a reconstituted system dependent on NADH and cytochrome b5 reductase, thus stimulating the activity of native cytochrome b5.     

Models for the complexes formed between cytochrome b5 and the subunits of methemoglobin

Computer graphics-generated models for the electron transfer complexes formed between cytochrome b5 and the subunits of methemoglobin are proposed. For both complexes, the orientation allowing optimal hydrogen bonding involves interaction between negatively charged residues on cytochrome b5 and positively charged residues on methemoglobin. In each complex, the heme groups of the interacting species are coplanar with the edges of the heme groups separated by 7-8 A and with the iron atoms 16 A apart. For the alpha-chain X cytochrome b5 complex, alpha-chain residues 56 (Lys), 60 (Lys), and 90 (Lys) interact with cytochrome b5 residues 44 (Glu), 43 (Glu), and 60 (Asp) respectively. A fourth hydrogen bond involves alpha-61 (Lys) bridging between a heme propionate from cytochrome b5 and a heme propionate from the alpha-chain. The contacts present in the beta-chain X cytochrome b5 complex involve hydrogen-bonding between beta-chain lysyl residues 59, 61, 65, and 95, and cytochrome b5 residues 48 (Glu), 44 (Glu), 43 (Glu), and 60 (Asp) respectively. An additional hydrogen bond can be formed by bridging of the epsilon-amino group of beta-66 (Lys) between a heme propionate from cytochrome b5 and a beta-chain heme propionate. In each complex, two nonionic interactions, one on each side of the heme groups, are also suggested. These interactions appear to effectively exclude external water molecules from the center of the protein-protein interaction domain. Comparison of the proposed binding loci for cytochrome b5 on the methemoglobin subunits with those proposed on cytochrome c reveals considerable structural homology between the cytochrome b5 binding sites.     

Diethyl pyrocarbonate modification abolishes fast electron accepting ability of cytochrome b561 from ascorbate but does not influence electron donation to monodehydroascorbate radical: identification of the modification sites by mass spectrometric analysis

Cytochrome b(561) from bovine adrenal chromaffin vesicles contains two heme B prosthetic groups and transports electron equivalents across the vesicle membranes to convert intravesicular monodehydroascorbate radical to ascorbate. To elucidate the mechanism of the transmembrane electron transfer, effects of the treatment of purified cytochrome b(561) with diethyl pyrocarbonate, a reagent specific for histidyl residues, were examined. We found that when ascorbate was added to the oxidized form of diethyl pyrocarbonate-treated cytochrome b(561), less than half of the heme iron was reduced but with a very slow rate. In contrast, radiolytically generated monodehydroascorbate radical was oxidized rapidly by the reduced form of diethyl pyrocarbonate-modified cytochrome b(561), as observed for untreated cytochrome b(561). These results indicate that the heme center specific for the electron acceptance from ascorbate was perturbed by the modification of amino acid residues nearby. We identified the major modification sites by mass spectrometry as Lys85, His88, and His161, all of which are fully conserved and located on the extravesicular side of cytochrome b(561) in the membranes. We suggest that specific N-carbethoxylation of the histidyl ligands of the heme b at extravesicular side abolishes the electron-accepting ability from ascorbate.     

Topological analysis of the active sites of cytochromes P450IIB4 (rabbit), P450IIB10 (mouse), and P450IIB11 (dog) by in situ rearrangement of phenyl-iron complexes.

The reaction of phenyldiazene with purified, phenobarbital-inducible rabbit cytochrome P450IIB4, mouse cytochrome P450IIB10, and dog cytochrome P450IIB11 yields complexes with absorbance maxima at 480 nm. Treatment of the cytochrome P450 complexes with K3Fe(CN)6 results in disappearance of the 480-nm absorption. Extraction of the prosthetic group from the proteins after these reactions yields the two isomers of N-phenylprotoporphyrin IX with the N-phenyl group on pyrrole rings A and D as the major products and the regioisomer with the N-phenyl on pyrrole ring C as a minor product. The A:C:D arylated pyrrole ring ratio is 3:2:3 for rabbit P450IIB4, 3:1:3 for mouse P450IIB10, and 4:1:2 for dog P450IIB11. Formation of the A and D regioisomers is consistent with the results obtained previously for rat isozymes IA1, IIB1, IIB2, and IIE1, but the rabbit, mouse, and dog P450IIB enzymes differ from the four rat enzymes in that a substantial amount of the isomer with the N-phenyl on pyrrole ring C is also formed. The results indicate that the region over pyrrole ring B is masked by protein residues in all the active sites and suggest that the region over pyrrole ring C is more hindered by protein residues in the rat than in the rabbit, mouse, or dog enzymes so far examined.     

Cytochrome spectrum of an obligate anaerobe, Eubacterium lentum.

An obligately anaerobic bacterium, Eubacterium lentum, was shown to contain cytochromes a, b, and c and a carbon monoxide-binding pigment. Extracts of cells grown with hemin gave a typical absorption spectrum for cytochrome c with maxima at 424, 525, and 553 nm. Extracts from cells grown in the absence of hemin also had an absorption peak corresponding to cytochrome b (562 nm) in their reduced versus oxidized spectrum. Extraction of hemes and formation of pyridine hemochromes allowed quantitation of protoheme IX and heme c. Large amounts of cytochrome c masked the presence of cytochrome b in cells grown in medium containing hemin. When cells were grown in the presence of 50 mM nitrate, cytochrome A (606 nm) was detected. In anaerobic extracts of cells grown either with or without nitrate, cytochromes b and c were reduced by formate and oxidized by NO3. Cytochrome a appeared to be partially oxidized by NO3 and completely oxidized by air.     

Sequence-specific 1H and 15N resonance assignments for both equilibrium forms of the soluble heme binding domain of rat ferrocytochrome b5.

15N and 1H resonance assignments for backbone and side-chain resonances of both equilibrium forms of rat ferrocytochrome b5 have been obtained, using 15N-1H heteronuclear correlation methods employing globally 15N-labeled protein. Unlike other cytochrome b5 species assigned to date (Guiles et al., 1990) the rat cytochrome exists as an equilibrium distribution of conformers in nearly equal abundance (Lee et al., 1990). The ratio of conformers present in all other species variants is approximately 1:9. More than 40% of all residues of the rat protein exhibit NMR-detectable heterogeneity due to the 180 degrees rotation of the heme about the alpha, gamma-meso axis. NOESY and HOHAHA relayed 15N-1H double-DEPT heteronuclear correlation methods were an indispensible tool for the deconvolution of a system with this level of heterogeneity. Differences in the resonance assignments between the two equilibrium conformers were found to be as great as differences between species variants we have previously reported. On the basis of the magnitude and extent of the observed chemical shift differences and specific NOESY connectivities observed in the two isomers, we believe the two equilibrium conformers differ not only by a simple back-to-front flip of the heme but also by an additional rotation about an axis normal to the heme plane as has been previously suggested by Pochapsky et al. (1990). A short segment of the protein at the N-terminus could not be assigned, presumably due to rapid exchange of solvent-accessible amide protons in this disordered segment of the protein. Assignments for 93 of the 98 residues of this 12-kDa protein have been obtained.     

(15)N backbone dynamics of ferricytochrome b(562): comparison with the reduced protein and the R98C variant.

The backbone dynamics of ferricytochrome b(562), a four-helix bundle protein from Escherichia coli, have been studied by NMR spectroscopy. The consequences of the introduction of a c-type thioether linkage between the heme and protein and the reduction to the ferrous cytochrome have also been analyzed. (15)N relaxation rates R(1) and R(2) and (1)H-(15)N NOEs were measured at proton Larmor frequencies of 500 and 600 MHz for the oxidized and reduced protein as well as for the oxidized R98C variant. In the latter protein, an "artificial" thioether covalent bond has been introduced between the heme group and the protein frame [Arnesano, F., Banci, L., Bertini, I., Ciofi-Baffoni, S., de Lumley Woodyear, T., Johnson, C. M., and Barker, P. D. (2000) Biochemistry 39, 1499-1514]. The (15)N relaxation data were analyzed with the ModelFree protocol, and the mobility parameters on the picosecond to nanosecond time scale were compared for the three species. The three forms are rather rigid as a whole, with average generalized order parameters values of 0.87 +/- 0.08 (oxidized cytochrome b(562)), 0.84 +/- 0.07 (reduced cytochrome b(562)), and 0.85 +/- 0.07 (oxidized R98C cytochrome b(562)), indicating similar mobility for each system. Lower order parameters (S(2)) are found for residues belonging to loops 1 and 2. Higher mobility, as indicated by lower order parameters, is found for heme binding helices alpha 1 and alpha 4 in the R98C variant with respect to the wild-type protein. The analysis requires a relatively long rotational correlation time (tau(m) = 9.6 ns) whose value is accounted for on the basis of the anisotropy of the molecular shape and the high phosphate concentration needed to ensure the occurrence of monomer species. A parallel study of motions in the millisecond to microsecond time scale has also been performed on oxidized wild-type and R98C cytochrome b(562). In a CPMG experiment, decay rates were analyzed in the presence of spin-echo pulse trains of variable spacing. The dynamic behavior on this time scale is similar to that observed on the sub-nanosecond time scale, showing an increased mobility in the residues connected to the heme ligands in the R98C variant. It appears that the increased protein stability of the variant, established previously, is not correlated with an increase in rigidity.     

A proton-NMR investigation of the fully reduced cytochrome c7 from Desulfuromonas acetoxidans. Comparison between the reduced and the oxidized forms.

The solution structure via 1H NMR of the fully reduced form of cytochrome c7 has been obtained. The protein sample was kept reduced by addition of catalytic amounts of Desulfovibrio gigas iron hydrogenase in H2 atmosphere after it had been checked that the presence of the hydrogenase did not affect the NMR spectrum. A final family of 35 conformers with rmsd values with respect to the mean structure of 8.7 +/- 1.5 nm and 12.4 +/- 1.3 nm for the backbone and heavy atoms, respectively, was obtained. A highly disordered loop involving residues 54-61 is present. If this loop is ignored, the rmsd values are 6.2 +/- 1.1 nm and 10.2 +/- 1.0 nm for the backbone and heavy atoms, respectively, which represent a reasonable resolution. The structure was analyzed and compared with the already available structure of the fully oxidized protein. Within the indetermination of the two solution structures, the result for the two redox forms is quite similar, confirming the special structural features of the three-heme cluster. A useful comparison can be made with the available crystal structures of cytochromes c3, which appear to be highly homologous except for the presence of a further heme. Finally, an analysis of the factors affecting the reduction potentials of the heme irons was performed, revealing the importance of net charges in differentiating the reduction potential when the other parameters are kept constant.     

1H-NMR assignments and the dynamics of interconversion of the isomeric forms of cytochrome b5 in solution

Cytochrome b5 reconstituted with specifically deuterated hemins has led to the assignment of the resolved 6,7 beta-propionate protons and heme meso protons. Freshly reconstituted cytochrome b5 contains a mixture of two isomers in an approx. 1:1 ratio. As time proceeds the minor isomer decreases in intensity until the equilibrium ratio, approx. 8:1, of the two isomers is reached. The rate of the heme disorder kinetics was investigated for cytochrome b5 as a function of pH, oxidation state and 2,4 heme substitutents. Comparison of the kinetic data for cytochrome b5 with that obtained for other b-type heme proteins supports the proposal that the heme disorder arises from a 180 degree rotation of the heme about the alpha, gamma-meso axis. Computer-difference methods allow the spectra of the two individual isomers to be generated. Comparison of the NMR spectral parameters for the two individual isomers indicates small structural differences for amino acid side-chain orientations.     

On the mechanism of action of cytochrome P-450. Oxidation and reduction of the ferrous dioxygen complex of liver microsomal cytochrome P-450 by cytochrome b5

The effects of cytochrome b5 on the decay of the ferrous dioxygen complexes of P-450LM2 and P-450LM4 from rabbit liver microsomes were studied by stopped-flow spectrophotometry. The P-450 (FeIIO2) complexes accept an electron from reduced cytochrome b5 and, in a reaction not previously described, donate an electron to oxidized cytochrome b5 to give ferric P-450. A comparison with the electron-transferring properties of ferrous P-450 under anaerobic conditions allowed determination of the limiting steps of the two reactions involving the oxygenated complex. The rate of decay of the dioxygen complex was increased in all cases with b5 present; however, with oxidized b5 a large increase in the rate was observed with P-450 isozyme 4 but not with isozyme 2, whereas the opposite situation was found when reduced b5 was used. The reactions between b5 and ferrous dioxygen P-450 were not at thermodynamic equilibrium under the conditions employed. From the results obtained, a model is proposed in which the ferrous dioxygen complex decomposes rapidly into another species differing from ferric P-450 in its spectral properties and from the starting complex in its electron-transferring properties. A scheme is presented to indicate how competition among spontaneous decay, cytochrome b5 oxidation, and cytochrome b5 reduction by the ferrous O2 complex may influence substrate hydroxylation.     

Binding of homogeneous cytochrome b5 to rat liver microsomes. Effect on N-demethylation reactions.

Incubation of rat homogeneous detergent-solubilized cytochrome b5 with rat liver microsomes resulted in specific binding of the hemoprotein which was rapidly reduced by NADH. The NADH cytochrome c reductase activity in these preparations increased in proportion to the amount of cytochrome bound. However, the extra-bound detergent-solubilized cytochrome b5 did inhibit NADPH-dependent N-demethylations, the NADH synergism and NADPH cytochrome P-450 reductase activity. Manganese protoporphyrin-apocytochrome complex when bound to microsomes in amounts equivalent to detergent-solubilised cytochrome b5 showed no effect on N-demethylation activity. Furthermore, the binding of cytochrome b5 preparations reconstituted from heme and apocytochrome b5 had no effect on either the NADPH-dependent N-demethylation of aminopyrene or ethylmorphine or the NADH synergism observed with rat liver microsomes. In addition, homogeneous cytochrome b5 eluted from three additional Sephadex G-100 columns showed no inhibitory effects when bound to liver microsomes. Spectral analyses of the acid-acetone extract of the hemoprotein showed an absorption peak at 278 nm suggesting that the homogeneous b5 contains contaminating amounts of tightly bound detergent which is responsible for the observed inhibition of mixed function oxidase activity and which is removed during extraction of the heme from the apocytochrome and during further gel filtration applications.     

Properties of electrophoretically homogeneous phenobarbital-inducible and beta-naphthoflavone-inducible forms of liver microsomal cytochrome P-450.

Procedures are described for the isolation of two forms of rabbit liver microsomal liver microsomal cytochrome P-450 (P-450LM) in homogeneous state. They are designated by their relative electrophoretic mobilities on polyacrylamide gel in the presence of sodium dodecyl sulfate as P-450LM2 and P-450LM4. P-450LM2, which was isolated from phenobarbital-induced animals, has a subunit molecular weight of 48,700. The best preparations contain 20 nmol of the cytochrome per mg of protein and 1 molecule of heme per polypeptide chain. P-450LM4, which is induced by beta-naphthoflavone but is also present in phenobarbital-induced and untreated animals, was isolated from all three sources and found to have a subunit molecular weight of 55,300. The best preparations contain 17nmol of the cytochrome per mg of protein and 1 molecule of heme per polypeptide chain. Some of the purified preparations of the cytochromes, although electrophoretically homogeneous, contain apoenzyme due to heme loss during purification. The purified proteins contain no detectable NADPH-cytochrome P-450 reductase, cytochrome b5, or NADH-cytochrome b5 reductase, and only low levels of phospholipid (about 1 molecule per subunit). Amino acid analysis indicated that P-450LM2 and P-450LM4 are similar in composition, but the latter protein has about 60 additional residues. The COOH-terminal amino acid of P-450LM2 is arginine, as shown by carboxypeptidase treatment, whereas that of P-450LM4 is lysine. NH2-terminal amino acid residues could not be detected. Carbohydrate analysis indicated that both cytochromes contain 1 residue of glucosamine and 2 of mannose per polypeptide subunit. The optical spectra of the oxidized and reduced cytochromes and carbon monoxide complexes were determined. Oxidized P-450LM2 has maxima at 568, 535, and 418 nm characteristic of a low spin hemeprotein, and P450LM4 from beta-naphthoflavone-induced, phenobarbital-induced, or control microsomes has maxima at 645 and 394 nm, characteristic of the high spin state. The spectrum of -450lm4 becomes similar to that of P-450LM2 at high protein concentrations or upon the addition of detergent (Renex), whereas the spectrum of P-450LM2 is unaffected by the protein concentration or the presence of detergent. Electron paramagnetic resonance spectrometry of the purified cytochromes indicated that oxidized -450lm2 is in the low spin state, whereas P-450LM4 is largely, but not entirely, in the high spin state.     

Monitoring mobility in the early steps of unfolding: the case of oxidized cytochrome b(5) in the presence of 2 M guanidinium chloride

A Model-Free analysis of the (15)N relaxation properties of oxidized cytochrome b(5), a heme-containing electron-transfer protein, has been performed in 2 M guanidinium chloride (GdmCl), i.e., just before the heme is released by the action of denaturant. This analysis provides information on the mobility in the nano- to picosecond time range. A parallel study on the motions in the milli- to microsecond time scale has also been performed by analyzing rotating-frame (15)N relaxation rates. The protein contains a 60:40 ratio of two conformers (A and B) differing for the rotation of the heme group around the alpha-gamma meso axis. The effect of denaturant has been followed for both species, and the mobility properties have been compared with the analogous information in the absence of denaturant. To complete the picture, we also performed (15)N relaxation measurements and the Model-Free analysis of the native B form, whereas data on the A form [Dangi, B., Sarma, S., Yan, C., Banville, D. L., Guiles, R. D. (1998) J. Phys. Chem. B 102, 8201-8208], as well as rotating-frame measurements for both native forms [Banci, L., Bertini, I., Cavazza, C., Felli, I. C., Koulougliotis, D. (1998) Biochemistry 37, 12320-12330; Arnesano, F., Banci, L., Bertini, I., Felli, I. C., Koulougliotis, D. (1999) Eur. J. Biochem. 260, 347-354], are already available in the literature. It is found that GdmCl tends to increase the internal mobility, although some residues are rigidified on both time scales. In the milli- to microsecond time scale, the tendency to increased mobility is reflected in a decrease in the tau(ex) values rather than in the number of residues experiencing conformational equilibria. In the nano- to picosecond time scale, the tendency to increased mobility is indicated by an overall decrease in the S(2) values. Color pictures are reported to visually show these effects. On the fast time scale, the B form is more mobile than the A form, reflecting the different stability with respect to unfolding. The increase in mobility upon addition of denaturant largely occurs around the heme pocket, which facilitates the release of the heme. The relevance of the internal motions with respect to the early steps of the unfolding process is also analyzed and discussed.     

Isolation and partial characterization of a new soluble b-type cytochrome (b9) from rat liver.

A new soluble cytochrome, designated as cytochrome b9, was purified to apparent homogeneity from rat liver. The absorption maximum of the oxidized (the native form) cytochrome b9 at room temperature was 413 nm. The dithionite-reduced cytochrome b9 had absorption maxima at 556, 527, and 423 nm. The prosthetic group of cytochrome b9 was identified as protoheme IX. From gel filtration experiments, the molecular weight of cytochrome b9 was estimated to be 125,000. Polyacrylamide gel electrophoresis experiments in the presence of sodium dodecyl sulfate showed that the molecular weight of its subunit was 61,000. The native form of cytochrome b9 was thus a dimer. The amount of heme/mol of dimer was 3.3 mol. Cytochrome b9 was autoxidizable and did not bind CO, 2.2 mM cyanide, or 2.2 mM azide. On the basis of its molecular weight of 125,000, the millimolar extinction coefficients of dimeric cytochrome b9 at 280 and 413 nm were 384 and 380, respectively. The absorbance at 280 nm/mg cytochrome b9 was 3.1. Cytochromes b9 and H-450 (I.-C. Kim and W.C. Deal (1976) Biochemistry 15, 4925-4930) are the only b-type, soluble cytochromes which have been isolated from mammalian liver; they are not found in tissues of heart, lung, kidney, and brain. The biological function of cytochrome b9 was not determined.     

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.     

Purification and properties of a diheme cytochrome b561 of the Escherichia coli respiratory chain

A new b-type cytochrome, cytochrome b561 (Murakami, H., Kita, K., Oya, H., and Anraku, Y. (1984) Mol. Gen. Genet. 196, 1-5) was purified to near homogeneity from the cytochrome b561-amplified Escherichia coli K12 strain HM204/pAM5029. The purified cytochrome b561 was a single polypeptide with a molecular weight of 18,000, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its isoelectric point was determined to be 9.6. The difference spectrum of the cytochrome at 77 K shows a major alpha-absorption peak at 561 nm and a minor peak at 555 nm. The absolute spectrum at room temperature of the oxidized form of the cytochrome had an absorption peak at 414 nm, and that of the reduced form had peaks at 562, 530, and 428 nm. The oxidation-reduction potential of the cytochrome was estimated to be +20 mV. The cytochrome contained 91.2 nmol of heme/mg of protein, showing that it was a cytoplasmic membrane-bound, b-type diheme cytochrome.     

Isolation and partial characterization of the cytochrome c oxidase of Micrococcus luteus (lysodeikticus)

The cell membrane of Micrococcus luteus (lysodeikticus) contains a respiratory chain composed of hemes a, b, and c, which contain 171, 457, and 407 pmol/mg protein, respectively. Cytochrome c oxidase, the heme a containing component, has been purified after solubilization in Triton X-100, by gel filtration on Sepharose 4B-CL ammonium sulfate precipitation and ion-exchange and affinity chromatographies on a yeast cytochrome c-Sepharose 4B column. The purified complex, which contains three polypeptides of apparent Mr 47,000, 31,000, and 19,000, has CN-sensitive ferrocytochrome c oxidase activity (Ki = 0.35 microM) and a characteristic absorption spectrum with maxima in the oxidized form at 595 and 426 nm and in the reduced form at 601 and 444 nm. The purified enzyme contains 17.4 nmol/mg protein and its copper content is 23.2 nmol/mg protein. The enzyme was purified about 100-fold with respect to its content in crude membranes. The total heme a yield, also with respect to crude membranes content, was 6.8%.