Probing the differences between rat liver outer mitochondrial membrane cytochrome b5 and microsomal cytochromes b5

Two distinct forms of cytochrome b5 exist in the rat hepatocyte. One is associated with the membrane of the endoplasmic reticulum (microsomal, or Mc, cyt b5) while the other is associated with the outer membrane of liver mitochondria (OM cyt b5). Rat OM cyt b5, the only OM cyt b5 identified so far, has a significantly more negative reduction potential and is substantially more stable toward chemical and thermal denaturation than Mc cytochromes b5. In addition, hemin is kinetically trapped in rat OM cyt b5 but not in the Mc proteins. As a result, no transfer of hemin from rat OM cyt b5 to apomyoglobin is observed at pH values as low as 5.2, nor can the thermodyamically favored ratio of hemin orientational isomers be achieved under physiologically relevant conditions. These differences are striking given the similarity of the respective protein folds. A combined theoretical and experimental study has been conducted in order to probe the structural basis behind the remarkably different properties of rat OM and Mc cytochromes b5. Molecular dynamics (MD) simulations starting from the crystal structure of bovine Mc cyt b5 revealed a conformational change that exposes several internal residues to the aqueous environment. The new conformation is equivalent to the "cleft-opened" intermediate observed in a previously reported MD simulation of bovine Mc cyt b5 [Storch, E. M., and Daggett, V. (1995) Biochemistry 34, 9682-9693]. The rat OM protein does not adopt a comparable conformation in MD simulations, thus restricting access of water to the protein interior. Subsequent comparisons of the protein sequences and structures suggested that an extended hydrophobic network encompassing the side chains of Ala-18, Ile-32, Leu-36, and Leu-47 might contribute to the inability of rat OM cyt b5 to adopt the cleft-opened conformation and, hence, stabilize its fold relative to the Mc isoforms. A corresponding network is not present in bovine Mc cyt b5 because positions 18, 32, and 47, are occupied by Ser, Leu, and Arg, respectively. To probe the roles played by Ala-18, Ile-32, and Leu-47 in endowing rat OM cyt b5 with its unusual structural properties, we have replaced them with the corresponding residues in bovine Mc cyt b5. Hence, the I32L (single), A18S/L47R (double), and A18S/L47R/I32L (triple) mutants of rat OM cyt b5 were prepared. The stability of these proteins was found to decrease in the following order: WT rat OM > rat OM I32L > rat OM A18S/L47R > rat OM A18S/L47R/I32L > bovine Mc cyt b5. The decrease in stability of the rat OM protein correlates with the extent to which the hydrophobic cluster involving the side chains of residues 18, 32, 36, and 47 has been disrupted. Complete disruption of the hydrophobic network in the triple mutant is confirmed in a 2.0 A resolution crystal structure of the protein. Disruption of the hydrophobic network also facilitates hemin loss at pH 5.2 for the double and triple mutants, with the less stable triple mutant exhibiting the greater rate of hemin transfer to apomyoglobin. Finally, 1H NMR spectroscopy and side-by-side comparisons of the crystal structures of bovine Mc, rat OM, and rat OM A18S/L47R/I32L cyt b5 allowed us to conclude that the nature of residue 32 plays a key role in controlling the relative stability of hemin orientational isomers A and B in rat OM cyt b5. A similar analysis led to the conclusion that Leu-70 and Ser-71 play a pivotal role in stabilizing isomer A relative to isomer B in Mc cytochromes b5.     

Toward engineering the stability and hemin-binding properties of microsomal cytochromes b5 into rat outer mitochondrial membrane cytochrome b5: examining the influence of residues 25 and 71

As part of a larger effort to engineer the stability and hemin-binding properties of microsomal (Mc) cytochromes b(5) into rat liver outer mitochondrial membrane (OM) cytochrome (cyt) b(5), several mutants of rat OM cyt b(5) were prepared to study the effect of gradual and complete elimination of two extended hydrophobic networks, which are present in the structure of the mitochondrial protein and are absent in the structure of mammalian Mc cytochromes b(5). One of the hydrophobic networks, identified in a previous study [Altuve, A., Silchenko, S., Lee, K.-H., Kuczera, K., Terzyan, S., Zhang, X., Benson, D. R., and Rivera, M. (2001) Biochemistry 40, 9469-9483], encompasses the side chains of Ala-18, Ile-32, Leu-36, and Leu-47, whereas a second hydrophobic network, identified as part of this work, encompasses the side chains of Ile-25, Phe-58, Leu-71, and the heme. The X-ray structure of the A18S/I25L/I32L/L47R/L71S quintuple mutant of rat OM cyt b(5) demonstrates that both hydrophobic networks have been eliminated and that the corresponding structural elements of the Mc isoform have been introduced. The stability of the rat OM mutant proteins studied was found to decrease in the order wild type > I25L > A18S/I32L/L47R > L71S > A18S/I32L/L47R/L71S > 18S/I25L/I32L/L47R/L71S, indicating that the two hydrophobic networks do indeed contribute to the high stability of rat OM cyt b(5) relative to the bovine Mc isoform. Surprisingly, the quintuple mutant of rat OM cyt b(5) is less stable than bovine Mc cyt b(5), even though the former exhibits significantly slower rates of hemin release and hemin reorientation at pH 7.0. However, at pH 5.0 the bovine Mc and rat OM quintuple mutant proteins release hemin at comparable rates, suggesting that one or both of the His axial ligands in the rat OM protein are more resistant to protonation under physiological conditions. Results obtained from chemical denaturation experiments conducted with the apoproteins demonstrated that mutants containing L71S are significantly less stable than bovine Mc apocyt b(5), strongly suggesting that Leu-71 plays a pivotal role in the stabilization of rat OM apocyt b(5), presumably via hydrophobic interactions with Ile-25 and Phe-58. Because comparable interactions are absent in bovine Mc apocyt b(5), which contains Ser at position 71, it must resort to different interactions to stabilize its fold, thus highlighting yet another difference between rat OM and bovine Mc cyt b(5). During the course of these investigations we also discovered that rat OM cyt b(5) can be made to strongly favor hemin orientational isomer A (I32L) or isomer B (L71S) with a single point mutation and that release of hemin orientational isomers A and B can be kinetically resolved in certain rat OM mutants.     

House fly cytochrome b5 exhibits kinetically trapped hemin and selectivity in hemin binding

We report that cytochrome b(5) (cyt b(5)) from Musca domestica (house fly) is more thermally stable than all other microsomal (Mc) cytochromes b(5) that have been examined to date. It also exhibits a much higher barrier to equilibration of the two isomeric forms of the protein, which differ by a 180 degrees rotation about the alpha-gamma-meso axis of hemin (ferric heme). In fact, hemin is kinetically trapped in a nearly statistical 1.2:1 ratio of rotational forms in freshly expressed protein. The equilibrium ratio (5.5:1) is established only upon incubation at temperatures above 37 degrees C. House fly Mc cyt b(5) is only the second b-hemoprotein that has been shown to exhibit kinetically trapped hemin at room temperature or above, the first being cyt b(5) from the outer membrane of rat liver mitochondria (rat OM cyt b(5)). Finally, we show that the small excess of one orientational isomer over the other in freshly expressed protein results from selective binding of hemin by the apoprotein, a phenomenon that has not heretofore been established for any apocyt b(5).     

Molecular dynamics simulation studies of cytochrome b5 from outer mitochondrial and microsomal membrane

Two forms of cytochrome b(5) have been identified, associated with the outer membrane of liver mitochondria (OM cyt b(5)) and with the membrane of the endoplasmic reticulum (microsomal, Mc cyt b(5)). These proteins have very similar structures, but differ significantly in physical properties, with the OM cyt b(5) exhibiting a more negative reduction potential, higher stability, and stronger interactions with the heme. We perform molecular dynamics simulations to probe the structures and fluctuations of the two proteins in solution, to help explain the observed physical differences. We find that the structures of the two proteins, highly similar in the crystal, differ in position of a surface loop involving residues 49-51 in solution. Hydrophobic residues Ala-18, Ile-32, Leu-36, and Leu-47 tend to cluster together on the surface of rat OM cyt b(5), blocking water access to the protein interior. In bovine Mc cyt b(5), two of these positions, Ser-18 and Arg-47, are occupied by hydrophilic residues. This leads to breaking the hydrophobic cluster and allowing the protein to occupy a more open conformation. A measure of this structural transition is the opening of a cleft on the protein surface, which is 5 A wider in the OM cyt b(5) simulation compared to the Mc form. The OM protein also appears to have a more compact hydrophobic core in its beta-sheet region. These effects may be used to explain observed stability differences between the two proteins.     

Mitochondrial and microsomal ferric b5 cytochromes exhibit divergent conformational plasticity in the context of a common fold

Native-state hydrogen-deuterium exchange (HDX) monitored by NMR spectroscopy has been used to compare conformational plasticity in ferric rat liver outer mitochondrial membrane cytochrome b5 (rOM b5) and ferric bovine liver microsomal cytochrome b5 (bMc b5). Analysis of the data indicated that rOM b5 is the less conformationally flexible protein on the time scale probed by the HDX experiments. The data also suggest a likely contributor to the much higher kinetic barrier for the release of hemin from OM b5s in comparison to Mc b5s, a characteristic that may be to a large extent the source of their divergent functional properties. Specifically, the data indicate that conformational mobility within helices alpha4 and alpha5, which flank the loop harboring axial ligand His63, is considerably more restricted in rOM b5 than in bMc b5. The lower conformational flexibility of alpha4 and alpha5 in rOM b5 can reasonably be attributed to more extensive hydrophobic packing in that region of the protein, arising from two conserved side chain packing motifs in OM cytochrome b5s [Altuve, A., Wang, L., Benson, D. R., and Rivera, M. (2004) Biochem. Biophys. Res. Commun. 314, 602-609].     

A histidine/tryptophan pi-stacking interaction stabilizes the heme-independent folding core of microsomal apocytochrome b5 relative to that of mitochondrial apocytochrome b5

The outer mitochondrial membrane isoform of mammalian cytochrome b5 (OM b5) is considerably more stable than its microsomal counterpart (Mc b5), whereas the corresponding apoproteins (OM and Mc apo-b5) exhibit similar stability. OM and Mc apo-b5 are also similar in that their empty heme-binding pockets (core 1) are highly disordered but that the remainder of each apoprotein (core 2) displays substantial hololike structure. Core 1 residue 71 is leucine in all known mammalian OM b5's and serine in the corresponding Mc proteins. Replacing Leu-71 in rat OM (rOM) b5 with Ser has been shown to (1) decrease apoprotein thermodynamic stability by >2 kcal/mol and (2) extend conformational disorder beyond core 1 and into core 2, as evidenced in part by loss of a near-UV circular dichroism signal associated with the side chain of invariant residue Trp-22. Herein we report identification of a conserved Mc b5 core 2 packing motif that plays a key role in stabilizing apoprotein conformation in the vicinity of Trp-22, thereby compensating for the presence of Ser at position 71: a pi-stacking interaction between the side chains of Trp-22 and His-15 that is extended by hydrogen bonding between the side chains of His-15, Ser-20, and Glu-11. The corresponding conserved packing motif in OM b5's differs in having arginine at position 15 and glutamate at position 20. We also present evidence indicating that the conserved Mc b5 packing motif noted above contributes to the unusually extensive secondary structure exhibited by bovine Mc apo-b5 in the urea-denatured state.     

Stabilizing roles of residual structure in the empty heme binding pockets and unfolded states of microsomal and mitochondrial apocytochrome b5

The microsomal (Mc) and mitochondrial (OM) isoforms of mammalian cytochrome b5 are the products of different genes, which likely arose via duplication of a primordial gene and subsequent functional divergence. Despite sharing essentially identical folds, heme-polypeptide interactions are stronger in OM b5s than in Mc b5s due to the presence of two conserved patches of hydrophobic amino acid side chains in the OM heme binding pockets. This is of fundamental interest in terms of understanding heme protein structure-function relationships, because stronger heme-polypeptide interactions in OM b5s in comparison to Mc b5s may represent a key source of their more negative reduction potentials. Herein we provide evidence that interactions amongst the amino acid side chains contributing to the hydrophobic patches in rat OM (rOM) b5 persist when heme is removed, rendering the empty heme binding pocket of rOM apo-b5 more compact and less conformationally dynamic than that in bovine Mc (bMc) apo-b5. This may contribute to the stronger heme binding by OM apo-b5 by reducing the entropic penalty associated with polypeptide folding. We also show that when bMc apo-b5 unfolds it adopts a structure that is more compact and contains greater nonrandom secondary structure content than unfolded rOM apo-b5. We propose that a more robust beta-sheet in Mc apo-b5s compensates for the absence of the hydrophobic packing interactions that stabilize the heme binding pocket in OM apo-b5s.     

Influence of point mutations on the flexibility of cytochrome b5: molecular dynamics simulations of holoproteins

Two membrane-bound isoforms of cytochrome b5 have been identified in mammals, one associated with the outer mitochondrial membrane (OM b5) and the other with the endoplasmic reticulum (microsomal, or Mc b5). The soluble heme binding domains of OM and Mc b5 have highly similar three-dimensional structures but differ significantly in physical properties, with OM b5 exhibiting higher stability due to stronger heme association. In this study, we present results of 8.5-ns length molecular dynamics simulations for rat Mc b5, bovine Mc b5, and rat OM b5, as well as for two rat OM b5 mutants that were anticipated to exhibit properties intermediate between those of rat OM b5 and the two Mc proteins: the A18S/I32L/L47R triple mutant (OM3M) and the A18S/I25L/I32L/L47R/L71S quintuple mutant (OM5M). Analysis of the structure, fluctuations, and interactions showed that the five b5 variants used in this study differed in organization of their molecular surfaces and heme binding cores in a way that could be used to explain certain experimentally observed physical differences. Overall, our simulations provided qualitative microscopic explanations of many of the differences in physical properties between OM and Mc b5 and two mutants in terms of localized changes in structure and flexibility. They also reveal that opening of a surface cleft between hydrophobic cores 1 and 2 in bovine Mc b5, observed in two previously reported simulations (E. M. Storch and V. Daggett, Biochemistry, 1995, Vol. 34, pp. 9682-9693; A. Altuve, Biochemistry, 2001, Vol. 40, pp. 9469-9483), probably resulted from removal of crystal contacts and likely does not occur on the nanosecond time scale. Finally, the MD simulations of OM5M b5 verify that stability and dynamic properties of cytochrome b5 are remarkably resistant to mutations that dramatically alter the stability and structure of the apoprotein.     

Divergence in nonspecific hydrophobic packing interactions in the apo state, and its possible role in functional specialization of mitochondrial and microsomal cytochrome b5

The outer mitochondrial membrane isoform of mammalian cytochrome b(5) (OM b(5)) is distinguished from the microsomal isoform (Mc b(5)) by its considerably greater stability. In contrast, OM and Mc apocytochrome b(5) (apo-b(5)) exhibit similar thermodynamic stability. Contributing substantially to the greater stability of OM b(5) relative to that of Mc b(5) is the presence of Leu at position 71. Replacing Leu-71 in OM b(5) with the corresponding Mc b(5) residue (Ser) not only diminishes holoprotein stability but also markedly compromises apoprotein stability. The studies reported herein were undertaken to clarify the role played by Leu-71 in stabilizing OM b(5)s relative to Mc b(5)s, and were motivated by the possibility that stability is related to other differences in OM and Mc b(5) properties that are important for their specialized subcellular roles. The results of these studies show that Leu-71 plays an essential role in maintaining the structural integrity of the heme-independent folding core of OM apo-b(5) (core 2), despite its location in the disordered empty heme-binding pocket (core 1). The conformational integrity of core 2 in Mc apo-b(5)s is not similarly dependent on the presence of a hydrophobic residue at position 71, providing new evidence for evolution of compensating structural features not present in OM b(5)s. We propose that Leu-71 achieves its effect on OM apo-b(5) core 2 structure by participating in a nonspecific hydrophobic collapse of disordered core 1, templated by more conformationally restricted side chains of residues in the beta-sheet that separates the two cores. We hypothesize that this has the added effect of maintaining core 1 of OM apo-b(5)s in a state more compact than that which occurs in Mc apo-b(5)s, possibly contributing to stronger heme binding by limiting the number of non-native conformations that the empty heme-binding pocket can populate.     

Mammalian mitochondrial and microsomal cytochromes b(5) exhibit divergent structural and biophysical characteristics

The only outer mitochondrial membrane cytochrome b(5) examined to date, from rat (rOM b(5)), exhibits greater stability than known mammalian microsomal (Mc) isoforms, as well as a much higher kinetic barrier for hemin dissociation and a more negative reduction potential. A BlastP search of available databases using the protein sequence of rOM b(5) as template revealed entries for analogous proteins from human (hOM b(5)) and mouse (mOM b(5)). We prepared a synthetic gene coding for the heme-binding domain of hOM b(5), and expressed the protein to high levels. The hOM protein exhibits stability, hemin-binding, and redox properties similar to those of rOM b(5), suggesting that they are characteristic of the OM b(5) subfamily. The divergence in properties between the OM and Mc b(5) isoforms in mammals can be attributed, at least in part, to the presence of two extended hydrophobic patches in the former. The biophysical properties characteristic of the OM proteins may be important in facilitating the two functions proposed for them so far, reduction of ascorbate radical and stimulation of androgen synthesis.     

Microsomal NADH-cytochrome b5 reductase of bovine brain: purification and properties

Bovine brain microsomal NADH-cytochrome b5 (cyt. b5) reductase [EC 1.6.2.2] was solubilized by digestion with lysosomes, and purified 8,500-fold with a 20% recovery by procedures including affinity chromatography on 5'-AMP-Sepharose 4B. The purified enzyme showed one band of a molecular weight of 31,000 on polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS). Polyacrylamide gel electrophoresis of the purified enzyme without SDS revealed a major band with a faint minor band, both of which exhibited NADH-cyt. b5 reductase activity. The isoelectric points of these components were 6.0 (major) and 6.3 (minor). The apparent Km values of the purified enzyme for NADH and ferricyanide were 1.1 and 4.2 microM, respectively. The apparent Km value for cyt. b5 was 14.3 microM in 10 mM potassium phosphate buffer (pH 7.5). The apparent Vmax value was 1,190 mumol cyt. b5 reduced/min/mg of protein. The NADH-cyt. b5 reductase activity of the purified enzyme was inhibited by sulfhydryl inhibitors and flavin analogues. Inhibition by phosphate buffer or other inorganic salts of the enzyme activity of the purified enzyme was proved to be of the competitive type. These properties were similar to those of NADH-cyt. b5 reductase from bovine liver microsomes or rabbit erythrocytes, although the estimated enzyme content in brain was about one-twentieth of that in liver (per g wet tissue). An immunochemical study using an antibody to purified NADH-cyt. b5 reductase bovine liver microsomes indicated that NADH-cyt. b5 reductase from brain microsomes is immunologically identical to the liver microsomal enzyme.     

Comparison of cytochromes b5 from insects and vertebrates

We report a 1.55 A X-ray crystal structure of the heme-binding domain of cytochrome b(5) from Musca domestica (house fly; HF b(5)), and compare it with previously published structures of the heme-binding domains of bovine microsomal cytochrome b(5) (bMc b(5)) and rat outer mitochondrial membrane cytochrome b(5) (rOM b(5)). The structural comparison was done in the context of amino acid sequences of all known homologues of the proteins under study. We show that insect b(5)s contain an extended hydrophobic patch at the base of the heme binding pocket, similar to the one previously shown to stabilize mammalian OM b(5)s relative to their Mc counterparts. The hydrophobic patch in insects includes a residue with a bulky hydrophobic side chain at position 71 (Met). Replacing Met71 in HF b(5) with Ser, the corresponding residue in all known mammalian Mc b(5)s, is found to substantially destabilize the holoprotein. The destabilization is a consequence of two related factors: (1) a large decrease in apoprotein stability and (2) extension of conformational disruption in the apoprotein beyond the empty heme binding pocket (core 1) and into the heme-independent folding core (core 2). Analogous changes have previously been shown to accompany replacement of Leu71 in rOM b(5) with Ser. That the stabilizing role of Met71 in HF b(5) is manifested primarily in the apo state is highlighted by the fact that its crystallographic Calpha B factor is modestly larger than that of Ser71 in bMc b(5), indicating that it slightly destabilizes local polypeptide conformation when heme is in its binding pocket. Finally, we show that the final unit of secondary structure in the cytochrome b(5) heme-binding domain, a 3(10) helix known as alpha6, differs substantially in length and packing interactions not only for different protein isoforms but also for given isoforms from different species.     

Direct electrochemistry of proteins. Investigations of yeast cytochrome c mutants and their complexes with cytochrome b5.

Direct electrochemistry of site-specific mutants of yeast iso-1-cytochrome c (cyt c) and their complexes with bovine cytochrome b5 (cyt b5) has been investigated at edge-plane pyrolytic graphite (EPG) and bis(4-pyridyl)-disulphide-modified gold electrodes. Structure/function relationships have been investigated with the particular aim of clarifying the factors controlling the interactions of proteins at electrode/electrolyte interfaces and the determinants for direct electrochemistry in ternary protein/protein/electrode adducts, e.g. cyt c/cyt b5/EPG. Investigations of the cyt c mutants alone revealed a variety of electrochemical responses: all the mutants show similar voltammetric reversibility at modified gold electrodes, whereas at EPG electrodes the reversibility follows the order: Asn52Ile-Cys102Thr greater than Cys102Thr greater than Asn52Ala-Cys102Thr. Mid-point potentials follow the order: Arg13Ile (+60 +/- 5 mV vs. standard calomel electrode) greater than Cys102Thr (+40 +/- 5 mV) greater than Lys27Gln (+30 +/- 5 mV) approximately Lys72Asp (+30 +/- 5 mV) greater than Asn52Ala-Cys102Thr (+15 +/- 5 mV) greater than Asn52Ile-Cys102Thr (-10 +/- 5 mV). The structural basis for these differences is briefly discussed. When these mutants are bound to cyt b5, the differences in electrochemical response are greatly enhanced in the ternary cyt c/cyt b5/EPG adducts. A minimal analysis of these differences supports a model of multiple overlapping binding and recognition domains on cyt c which may be finely tuned to allow ternary complex formation so that a single-site variation could modify or abolish direct electrochemistry in the ternary adduct.     

Expression of lipase-solubilized bovine liver microsomal cytochrome b5 in Escherichia coli as a glutathione S-transferase fusion protein (GST-cyt b5)

The gene coding for the lipase-solubilized bovine liver microsomal cytochrome b5 (cyt b5) was expressed in Escherichia coli BL21 cells as a glutathione S-transferase fusion protein (GST-cyt b5) using the constructed expression vector pGEX-cyt b). The GST-cyt b5 fusion protein can be matured in vivo as a holoprotein with heme incorporated into cyt b5 during the fermentation, and the purification procedures were simplified by using a one-step affinity column chromatography with glutathione-agarose gel. The fusion protein was characterized by its spectroscopic and electrochemical properties, the interaction between GST-cyt b5 and cyt c was also investigated. The results show that GST-cyt b5 fusion protein shares similar properties and functions to that of isolated cyt b5. Although cyt b5 and GST were fused together, the two partners have not made significant structural and functional alterations of their counterparts, the protein-protein interactions between them are apparently very weak. To our knowledge, the present study is the first report to express cyt b5 as a GST-cyt b5 fusion protein, which provides a good example for the in vivo maturation of a hemoprotein as a GST fusion protein and sheds new light on the protein-protein interactions within the GST fusion protein.     

Characterization and calculation of a cytochrome c-cytochrome b5 complex using NMR data

To identify the binding site for bovine cytochrome b(5) (cyt b(5)) on horse cytochrome c (cyt c), cross-saturation transfer NMR experiments were performed with (2)H- and (15)N-enriched cyt c and unlabeled cyt b(5). In addition, chemical shift changes of the cyt c backbone amide and side chain methyl resonances were monitored as a function of cyt b(5) concentration. The chemical shift changes indicate that the complex is in fast exchange, and are consistent with a 1:1 stoichiometry. A K(a) of (4 +/- 3) x 10(5) M(-)(1) was obtained with a lower limit of 855 s(-)(1) for the dissociation rate of the complex. Mapping of the chemical shift variations and intensity changes upon cross-saturation NMR experiments in the complex reveals a single, contiguous interaction interface on cyt c. Using NMR data as constraints, a protein docking program was used to calculate two low-energy model complex clusters. Independent calculations of the effect of the cyt b(5) heme ring current-induced magnetic dipole on cyt c were used to discriminate between the different models. The interaction surface of horse cyt c in the current experimentally constrained model of the cyt c-cyt b(5) complex is similar but not identical to the interface predicted in yeast cyt c by Brownian dynamics and docking calculations. The occurrence of different amino acids at the protein-protein interface and the dissimilar assumptions employed in the calculations can largely account for the nonidentical interfaces.     

Purification and characterization of bovine adrenal cytochrome b561 expressed in insect and yeast cell systems

Bovine adrenal chromaffin granule cytochrome (cyt) b561 is a transmembrane hemoprotein that plays a key role in transporting reducing equivalents from ascorbate to dopamine-beta-hydroxylase for catecholamine synthesis. We have developed procedures for expression and purification of functional bovine adrenal cyt b561 in insect and yeast cell systems. The bovine cyt b561 coding sequence, with or without a hexahistidine-tag sequence at the C-terminus, was cloned into the pVL1392 transfer vector under the control of the polyhedrin promoter to generate recombinant baculovirus for protein expression in Sf9 insect cells (approximately 0.5 mg detergent-solubilized cyt b561/L culture). For the yeast system, the cyt b561 cDNA was modified with a hexahistidine-tag sequence at the C-terminus, and inserted into the pPICZB vector under the control of the alcohol oxidase promoter. The recombinant plasmid was transformed into Pichia pastoris GS115 competent cells to give methanol-inducible cyt b561 expression (approximately 0.7 mg detergent-solubilized cyt b561/L culture). Recombinant His-tagged cyt b561 expressed in Sf9 or Pichia cells was readily solubilized from membrane fractions with dodecyl maltoside and purified to electrophoretic homogeneity by one-step chromatography on Ni-NTA affinity resin. The purified recombinant cytochrome from both systems had a heme to protein ratio close to two and was fully functional, as judged by comparison with the spectroscopic and kinetic parameters of the endogenous cytochrome from chromaffin granules. A novel procedure for isolation of chromaffin granule membranes was developed to utilize frozen adrenal glands instead of fresh tissue.     

Effect of famoxadone on photoinduced electron transfer between the iron-sulfur center and cytochrome c1 in the cytochrome bc1 complex

Famoxadone is a new cytochrome bc(1) Q(o) site inhibitor that immobilizes the iron-sulfur protein (ISP) in the b conformation. The effects of famoxadone on electron transfer between the iron-sulfur center (2Fe-2S) and cyt c(1) were studied using a ruthenium dimer to photoinitiate the reaction. The rate constant for electron transfer in the forward direction from 2Fe-2S to cyt c(1) was found to be 16,000 s(-1) in bovine cyt bc(1). Binding famoxadone decreased this rate constant to 1,480 s(-1), consistent with a decrease in mobility of the ISP. Reverse electron transfer from cyt c(1) to 2Fe-2S was found to be biphasic in bovine cyt bc(1) with rate constants of 90,000 and 7,300 s(-1). In the presence of famoxadone, reverse electron transfer was monophasic with a rate constant of 1,420 s(-1). It appears that the rate constants for the release of the oxidized and reduced ISP from the b conformation are the same in the presence of famoxadone. The effects of famoxadone binding on electron transfer were also studied in a series of Rhodobacter sphaeroides cyt bc(1) mutants involving residues at the interface between the Rieske protein and cyt c(1) and/or cyt b.     

Cytochrome b5 increases the rate of product formation by cytochrome P450 2B4 and competes with cytochrome P450 reductase for a binding site on cytochrome P450 2B4

The kinetics of product formation by cytochrome P450 2B4 were compared in the presence of cytochrome b(5) (cyt b(5)) and NADPH-cyt P450 reductase (CPR) under conditions in which cytochrome P450 (cyt P450) underwent a single catalytic cycle with two substrates, benzphetamine and cyclohexane. At a cyt P450:cyt b(5) molar ratio of 1:1 under single turnover conditions, cyt P450 2B4 catalyzes the oxidation of the substrates, benzphetamine and cyclohexane, with rate constants of 18 +/- 2 and 29 +/- 4.5 s(-1), respectively. Approximately 500 pmol of norbenzphetamine and 58 pmol of cyclohexanol were formed per nmol of cyt P450. In marked contrast, at a cyt P450:CPR molar ratio of 1:1, cyt P450 2B4 catalyzes the oxidation of benzphetamine congruent with100-fold (k = 0.15 +/- 0.05 s(-1)) and cyclohexane congruent with10-fold (k = 2.5 +/- 0.35 s(-1)) more slowly. Four hundred picomoles of norbenzphetamine and 21 pmol of cyclohexanol were formed per nmol of cyt P450. In the presence of equimolar concentrations of cyt P450, cyt b(5), and CPR, product formation is biphasic and occurs with fast and slow rate constants characteristic of catalysis by cyt b(5) and CPR. Increasing the concentration of cyt b(5) enhanced the amount of product formed by cyt b(5) while decreasing the amount of product generated by CPR. Under steady-state conditions at all cyt b(5):cyt P450 molar ratios examined, cyt b(5) inhibits the rate of NADPH consumption. Nevertheless, at low cyt b(5):cyt P450 molar ratios 相似文献   

Mapping the binding interface of the cytochrome b5-cytochrome c complex by nuclear magnetic resonance

The interaction between bovine cytochrome b(5) (cyt b(5)) and horse heart cytochrome c (cyt c) is investigated by NMR spectroscopy. Chemical shifts of cyt b(5) backbone resonances and side chain methyl resonances were monitored as a function of cyt c concentration. The shifts are small but saturatable and indicate that the binding of cyt b(5) with cyt c is in fast exchange. An equilibrium association constant of (6 +/- 3) x 10(4) M(-1) was obtained with a lower limit of 180 s(-1) for the dissociation rate of the complex. To resolve considerable ambiguities in the interpretation of the chemical shift mapping, (15)N relaxation experiments and cross-saturation experiments were used as alternative methods to map the cyt b(5)-cyt c binding interface. Results from the three experiments combined demonstrate that the conserved negatively charged region of cyt b(5) surrounding the solvent-exposed heme edge is involved in the interaction with cyt c. These data support the models proposed by Salemme and Mauk [(1976) J. Mol. Biol. 102, 563-568; (1993) Biochemistry 32, 6613-6623].     

Cytochrome b5 inhibits electron transfer from NADPH-cytochrome P450 reductase to ferric cytochrome P450 2B4

Experiments demonstrating that cytochrome (cyt) b5 inhibits the activity of cytochrome P450 2B4 (cyt P450 2B4) at higher concentrations suggested that cyt b5 was occupying the cyt P450 reductase-binding site on cyt P450 2B4 and preventing the reduction of ferric cyt P450 (Zhang, H., Im, S.-C., and Waskell, L. (2007) J. Biol. Chem. 282, 29766-29776). In this work experiments were undertaken with manganese-containing cyt b5 (Mn-cyt b5) to test this hypothesis. Because Mn-cyt b5 does not undergo oxidation state changes under our experimental conditions, interpretation of the experimental results was unambiguous. The rate of electron transfer from cyt P450 reductase to ferric cyt P450 2B4 was decreased by Mn-cyt b5 in a concentration-dependent manner. Moreover, reduction of cyt P450 2B4 by cyt P450 reductase was incomplete in the presence of Mn-cyt b5. At a Mn-cyt b(5):cyt P450 2B4:cyt P450 reductase molar ratio of 5:1:1, the rate of reduction of ferric cyt P450 was decreased by 10-fold, and only 30% of the cyt P450 was reduced, whereas 70% remained oxidized. It could be demonstrated that Mn-cyt b5 had its effect by acting on cyt P450, not the reductase, because the reduction of cyt c by cyt P450 reductase in the presence of Mn-cyt b5 was not effected. Furthermore, under steady-state conditions in the cyt P450 reconstituted system, Mn-cyt b5, which lacks the ability to reduce oxyferrous cyt P450 2B4, was unable to stimulate the activity of cyt P450. Mn-cyt b5 only inhibited the cyt P450 2B4 activity. In conjunction with site-directed mutagenesis studies and experiments that strongly suggested that cyt b5 competed with cyt P450 reductase for binding to cyt P450, the current investigation demonstrates unequivocally that cyt b5 inhibits the activity of cyt P450 2B4 by preventing cyt P450 reductase from binding to cyt P450, a prerequisite for electron transfer from cyt P450 reductase to cyt P450 and catalysis.