Arginine 91 is not essential for flavin incorporation in hepatic cytochrome b(5) reductase

Cytochrome b(5) reductase (cb5r) catalyzes the transfer of reducing equivalents from NADH to cytochrome b(5). Utilizing an efficient heterologous expression system that produces a histidine-tagged form of the hydrophilic, diaphorase domain of the enzyme, site-directed mutagenesis has been used to generate cb5r mutants with substitutions at position 91 in the primary sequence. Arginine 91 is an important residue in binding the FAD prosthetic group and part of a conserved "RxY(T)(S)xx(S)(N)" sequence motif that is omnipresent in the "ferredoxin:NADP(+) reductase" family of flavoproteins. Arginine 91 was replaced with K, L, A, P, D, Q, and H residues, respectively, and all the mutant proteins purified to homogeneity. Individual mutants were expressed with variable efficiency and all exhibited molecular masses of approximately 32 kDa. With the exception of R91H, all the mutants retained visible absorption spectra typical of a flavoprotein, the former being produced as an apoprotein. Visible absorption spectra of R91A, L, and P were red shifted with maxima at 458 nm, while CD spectra indicated an altered FAD environment for all the mutants except R91K. Fluorescence spectra showed a reduced degree of intrinsic flavin fluorescence quenching for the R91K, A, and P, mutants, while thermal stability studies suggested all the mutants, except R91K, were somewhat less stable than the wild-type domain. Initial-rate kinetic measurements demonstrated that the mutants exhibited decreased NADH:ferricyanide reductase activity with the R91P mutant retaining the lowest activity, corresponding to a k(cat) of 283 s(-1) and a K(NADH)(m) of 105 microM, when compared to the wild-type domain (k(cat) = 800 s(-1) K(NADH)(m) = 6 microM). These results demonstrate that R91 is not essential for FAD binding in cb5r; however, mutation of R91 perturbs the flavin environment and alters both diaphorase substrate recognition and utilization.     

Cytochrome b5 reductase: role of the si-face residues, proline 92 and tyrosine 93, in structure and catalysis

The conserved sequence motif "RxY(T)(S)xx(S)(N)" coordinates flavin binding in NADH:cytochrome b(5) reductase (cb(5)r) and other members of the flavin transhydrogenase superfamily of oxidoreductases. To investigate the roles of Y93, the third and only aromatic residue of the "RxY(T)(S)xx(S)(N)" motif, that stacks against the si-face of the flavin isoalloxazine ring, and P92, the second residue in the motif that is also in close proximity to the FAD moiety, a series of rat cb(5)r variants were produced with substitutions at either P92 or Y93, respectively. The proline mutants P92A, G, and S together with the tyrosine mutants Y93A, D, F, H, S, and W were recombinantly expressed in E. coli and purified to homogeneity. Each mutant protein was found to bind FAD in a 1:1 cofactor:protein stoichiometry while UV CD spectra suggested similar secondary structure organization among all nine variants. The tyrosine variants Y93A, D, F, H, and S exhibited varying degrees of blue-shift in the flavin visible absorption maxima while visible CD spectra of the Y93A, D, H, S, and W mutants exhibited similar blue-shifted maxima together with changes in absorption intensity. Intrinsic flavin fluorescence was quenched in the wild type, P92S and A, and Y93H and W mutants while Y93A, D, F, and S mutants exhibited increased fluorescence when compared to free FAD. The tyrosine variants Y93A, D, F, and S also exhibited greater thermolability of FAD binding. The specificity constant (k(cat)/K(m)(NADH)) for NADH:FR activity decreased in the order wild type > P92S > P92A > P92G > Y93F > Y93S > Y93A > Y93D > Y93H > Y93W with the Y93W variant retaining only 0.5% of wild-type efficiency. Both K(s)(H4NAD) and K(s)(NAD+) values suggested that Y93A, F, and W mutants had compromised NADH and NAD(+) binding. Thermodynamic measurements of the midpoint potential (E degrees ', n = 2) of the FAD/FADH(2) redox couple revealed that the potentials of the Y93A and S variants were approximately 30 mV more positive than that of wild-type cb(5)r (E degrees ' = -268 mV) while that of Y93H was approximately 30 mV more negative. These results indicate that neither P92 nor Y93 are critical for flavin incorporation in cb(5)r and that an aromatic side chain is not essential at position 93, but they demonstrate that Y93 forms contacts with the FAD that effectively modulate the spectroscopic, catalytic, and thermodynamic properties of the bound cofactor.     

Heterologous expression of an endogenous rat cytochrome b(5)/cytochrome b(5) reductase fusion protein: identification of histidines 62 and 85 as the heme axial ligands

The gene coding for expression of an endogenous soluble fusion protein comprising a b-type cytochrome-containing domain and a FAD-containing domain has been cloned from rat liver mRNA. The 1461-bp hemoflavoprotein gene corresponded to a protein of 493 residues with the heme- and FAD-containing domains comprising the amino and carboxy termini of the protein, respectively. Sequence analysis indicated the heme and flavin domains were directly analogous to the corresponding domains in microsomal cytochrome b(5) (cb5) and cytochrome b(5) reductase (cb5r), respectively. The full-length fusion protein was purified to homogeneity and demonstrated to contain both heme and FAD prosthetic groups by spectroscopic analyses and MALDI-TOF mass spectrometry. The cb5/cb5r fusion protein was able to utilize both NADPH and NADH as reductants and exhibited both NADPH:ferricyanide (k(cat) = 21.7 s(-1), K(NADPH)(m) = 1 microM. K(FeCN6)(m) = 8 microM) and NADPH:cytochrome c (k(cat) = 8.3 s(-1), K(NADPH)(m) = 1 microM. K(cyt c)(m) = 7 microM) reductase activities with a preference for NADPH as the reduced pyridine nucleotide substrate. NADPH-reduction was stereospecific for transfer of the 4R-proton and involved a hydride transfer mechanism with a kinetic isotope effect of 3.1 for NADPH/NADPD. Site-directed mutagenesis was used to examine the role of two conserved histidine residues, H62 and H85, in the heme domain segment. Substitution of either residue by alanine or methionine resulted in the production of simple flavoproteins that were effectively devoid of both heme and NAD(P)H:cytochrome c reductase activity while retaining NAD(P)H:ferricyanide activity, confirming that the former activity required a functional heme domain. These results have demonstrated that the rat cb5/cb5r fusion protein is homologous to the human variant and has identified the heme and FAD as the sites of interaction with cytochrome c and ferricyanide, respectively. Mutagenesis has confirmed the identity of both axial heme ligands which are equivalent to the corresponding residues in microsomal cytochrome b(5).     

Mutagenesis of Glycine 179 modulates both catalytic efficiency and reduced pyridine nucleotide specificity in cytochrome b5 reductase

Cytochrome b5 reductase (cb5r), a member of the ferredoxin:NADP+ reductase family of flavoprotein transhydrogenases, catalyzes the NADH-dependent reduction of cytochrome b5. Within this family, a conserved "GxGxxP" sequence motif has been implicated in binding reduced pyridine nucleotides. However, Glycine 179, a conserved residue in cb5r primary structures, precedes this six-residue "180GxGxxP185" motif that has been identified as binding the adenosine moiety of NADH. To investigate the role of G179 in NADH complex formation and NAD(P)H specificity, a series of rat cb5r variants were generated, corresponding to G179A, G179P, G179T, and G179V, recombinantly expressed in Escherichia coli and purified to homogeneity. Each mutant protein was found to incorporate FAD in a 1:1 cofactor/protein stoichiometry and exhibited absorption and CD spectra that were identical to those of wild-type cb5r, indicating both correct protein folding and similar flavin environments, while oxidation-reduction potentials for the FAD/FADH2 couple (n = 2) were also comparable to the wild-type protein (E(o)' = -272 mV). All four mutants showed decreased NADH:ferricyanide reductase activities, with kcat decreasing in the order WT > G179A > G179P > G179T > G179V, with the G179V variant retaining only 1.5% of the wild-type activity. The affinity for NADH also decreased in the order WT > G179A > G179P > G179T > G179V, with the Km(NADH) for G179V 180-fold greater than that of the wild type. Both Ks(H4NAD) and Ks(NAD+) values confirmed that the G179 mutants had both compromised NADH- and NAD+-binding affinities. Determination of the NADH/NADPH specificity constant for the various mutants indicated that G179 also participated in pyridine nucleotide selectivity, with the G179V variant preferring NADPH approximately 8000 times more than wild-type cb5r. These results demonstrated that, while G179 was not critical for either flavin incorporation or maintenance of the appropriate flavin environment in cb5r, G179 was required for both effective NADH/NADPH selectivity and to maintain the correct orientation and position of the conserved cysteine in the proline-rich "CGpppM" motif that is critical for optimum NADH binding and efficient hydride transfer.     

Expression and characterization of a functional canine variant of cytochrome b5 reductase

Cytochrome b5 reductase (cb5r), a member of the flavoprotein transhydrogenase family of oxidoreductase enzymes, catalyzes the transfer of reducing equivalents from the physiological electron donor, NADH, to two molecules of cytochrome b5. We have determined the correct nucleotide sequence for the putative full-length, membrane-associated enzyme from Canis familiaris, and have generated a heterologous expression system for production of a histidine-tagged variant of the soluble, catalytic diaphorase domain, comprising residues I33 to F300. Using a simple two-step chromatographic procedure, the recombinant diaphorase domain has been purified to homogeneity and demonstrated to be a simple flavoprotein with a molecular mass of 31,364 (m/z) that retained both NADH:ferricyanide reductase and NADH:cytochrome b5 reductase activities. The recombinant protein contained a full complement of FAD and exhibited absorption and CD spectra comparable to those of a recombinant form of the rat cytochrome b5 reductase diaphorase domain generated using an identical expression system, suggesting similar protein folding. Oxidation-reduction potentiometric titrations yielded a standard midpoint potential (Eo') for the FAD/FADH2 couple of -273+/-5 mV which was identical to the value obtained for the corresponding rat domain. Thermal denaturation studies revealed that the canine domain exhibited stability comparable to that of the rat protein, confirming similar protein conformations. Initial-rate kinetic studies revealed the canine diaphorase domain retained a marked preference for NADH versus NADPH as reducing substrate and exhibited kcat's of 767 and 600 s(-1) for NADH:ferricyanide reductase and NADH:cytochrome b5 reductase activities, respectively, with Km's of 7, 8, and 12 microM for NADH, K3Fe(CN)6, and cytochrome b5, respectively. Spectral-binding constants (Ks) determined for a variety of NAD+ analogs indicated the highest and lowest affinities were observed for APAD+ (Ks=71 microM) and PCA+ (Ks=>31 mM), respectively, and indicated the binding contributions of the various portions of the pyridine nucleotide. These results provide the first correct sequence for the full-length, membrane-associated form of C. familiaris cb5r and provide a direct comparison of the enzymes from two phylogenetic sources using identical expression systems that indicate that both enzymes have comparable spectroscopic, kinetic, thermodynamic, and structural properties.     

Expression of a novel P275L variant of NADH:cytochrome b5 reductase gives functional insight into the conserved motif important for pyridine nucleotide binding

The clinical disorder of recessive congenital methemoglobinemia (RCM, OMIN 250800) is associated with mutations in NADH:cytochrome b5 reductase (cb5r) and manifests as cyanosis from birth. Screening a cyanotic infant indicated elevated methemoglobin levels and decreased cb5r activity suggesting RCM. Sequencing the DIA1 gene encoding cb5r revealed a novel mutation, C27161T (NCBI accession number: NT_011520), resulting in replacement of proline at amino acid 275 with leucine (P275L). To understand how this mutation would affect cb5r's function, the P275L variant was expressed in a heterologous expression system and spectroscopic, thermodynamic, and thermostability studies were performed. The leucine substitution at residue 275 was found to significantly decrease the affinity towards the physiological reducing substrate, NADH, without affecting the activity of the P275L variant. From the rat model, residue 275 is predicted to be part of a conserved "CGPPPM" motif important for the binding and correct positioning of the NADH reducing substrate. Thus P275 influences the interaction with NADH which was confirmed by the change in affinity towards the physiological reducing substrate.     

Simultaneous purification and characterization of cytochrome b5 reductase and cytochrome b5 from sheep liver

Cytochrome b5 was purified from detergent solubilized sheep liver microsomes by using three successive DEAE-cellulose, and Sephadex G-100 column chromatographies. It was purified 54-fold and the yield was 23.5% with respect to microsomes. The apparent Mr of cytochrome b5 was estimated to be 16,200 +/- 500 by SDS-PAGE. Absolute absorption spectrum of the purified cytochrome b5 showed maximal absorption at 412 nm and dithionite-reduced cytochrome b5 gave peaks at 557, 526.5 and 423 nm. The ability of the purified sheep liver cytochrome b5 to transfer electrons from NADH-cytochrome b5 reductase to cytochrome c was investigated. The K(m) and Vmax values were calculated to be 0.088 microM cytochrome b5 and 315.8 microM cytochrome c reduced/min/mg enzyme, respectively. Also the reduction of cytochrome b5 by reductase was studied and K(m) and Vmax values were determined to be 5 microM cytochrome b5 and 5200 nmol cytochrome b5 reduced/min/mg enzyme, respectively. The K(m) and Vmax values for the cofactor NADH in the presence of saturating concentration of cytochrome b5 were found to be 0.0017 mM NADH and 6944 nmol cytochrome b5 reduced/min/mg enzyme, respectively. NADH-cytochrome b5 reductase was also partially purified from the same source, detergent solubilized sheep liver microsomes, by using two successive DEAE-cellulose, and 5'-ADP-agarose affinity column chromatographies. It was purified 144-fold and the yield was 7% with respect to microsomes. The apparent monomer Mr of reductase was estimated to be 34,000 by SDS-PAGE. When ferricyanide was used as an electron acceptor, reductase showed maximum activity between 6.8 and 7.5. The K(m) and Vmax values of the enzyme for ferricyanide were calculated as 0.024 mM ferricyanide and 673 mumol ferricyanide reduced/min/mg enzyme, respectively. The K(m) and Vmax values for the cofactor NADH in the presence of saturating amounts of ferricyanide were found to be 0.020 mM NADH and 699 mumol ferricyanide reduced/min/mg enzyme, respectively.     

Cytochrome b5 reductase: the roles of the recessive congenital methemoglobinemia mutants P144L, L148P, and R159*

Recessive congenital methemoglobinemia (RCM, OMIM 250800) arises from defects in either the erythrocytic or microsomal forms of the flavoprotein, cytochrome b5 reductase (cb5r) and was the first disease to be directly associated with a specific enzyme deficiency. Of the 33 verified mutations in cb5r that give rise to either the type I (erythrocytic) or type II (generalized) forms of RCM, three of the mutations, corresponding to P144L, L148P, and R159*, are located in a segment of the primary sequence composed of residues G143 to V171 which serves as a "hinge" or "linker" region between the FAD- and NADH-binding lobes of the protein. With the exception of R159*, which produces a truncated non-functional cb5r resulting in type II RCM, the type I methemoglobinemias resulting from the P144L or L148P mutations have been proposed to be due to decreased enzyme stability. Utilizing a recombinant form of the rat cb5r enzyme, we have generated the P144L, L148P, and P144L/L148P mutants, purified the resulting proteins to homogeneity and characterized their spectroscopic, kinetic, and thermodynamic properties. The three mutant proteins retained full complements of FAD with the P144L and L148P variants being spectroscopically indistinguishable from wild-type cb5r. In contrast, kinetic analyses revealed that the P144L, L148P, and P144L/L148P variants retained only 28, 31, and 8% of wild-type NADH:cytochrome b5 reductase activity, respectively, together with significant alterations in affinity for both NADH and NAD+. In addition, FAD oxidation-reduction potentials were 32, 19, and 65 mV more positive for the mutants than the corresponding FAD/FADH2 couple in native cb5r (E0'=-272 mV). Thermal and proteolytic stability measurements indicated that all three mutants were less stable than the wild-type protein while differential spectroscopy indicated altered pyridine nucleotide binding in all three variants. These results demonstrate that the "hinge" region is important in maintaining the correct orientation of the flavin- and pyridine nucleotide-binding lobes within the protein for efficient electron transfer and that the P144L and L148P mutations disrupt the normal registration of the FAD- and NADH-binding lobes resulting in altered affinities for both the physiological reducing substrate, NADH and its product, NAD+.     

Effects of flavin-binding motif amino acid mutations in the NADH-cytochrome b5 reductase catalytic domain on protein stability and catalysis

Porcine NADH-cytochrome b5 reductase catalytic domain (Pb5R) has the RXY(T/S)+(T/S) flavin-binding motif that is highly conserved among the structurally related family of flavoprotein reductases. Mutations were introduced that alter the Arg(63), Tyr(65), and Ser(99) residues within this motif. The mutation of Tyr(65) to either alanine or phenylalanine destabilized the protein, produced an accelerated release of FAD in the presence of 1.5 M guanidine hydrochloride, and decreased the k(cat) values of the enzyme. These results indicate that Tyr(65) contributes to the stability of the protein and is important in the electron transfer from NADH to FAD. The mutation of Ser(99) to either alanine or valine, and of Arg(63) to either alanine or glutamine increased both the K(m) values for NADH (K(m)(NADH)) and the dissociation constant for NAD(+) (K(d)(NAD+)). However, the mutation of Ser(99) to threonine and of Arg(63) to lysine had very little effect on the K(m)(NADH) and K(d)(NAD+) values, and resulted in small changes in the absorption and circular dichroism spectra. These results suggest that the hydroxyl group of Ser(99) and the positive charge of Arg(63) contribute to the maintenance of the properties of FAD and to the effective binding of Pb5R to both NADH and NAD(+). In addition, the mutation of Arg(63) to either alanine or glutamine increased the apparent K(m) values for porcine cytochrome b5 (Pb5), while changing Arg(63) to lysine did not. The positive charge of Arg(63) may regulate the electron transfer through the electrostatic interaction with Pb5. These results substantiate the important role of the flavin-binding motif in Pb5R.     

Improving protein extraction yield in reversed micellar systems through surface charge engineering

The mechanism of extraction of rat cytochrome b(5) from water into a sodium dioctylsulfosuccinate (AOT) micellar organic phase was studied using protein engineering of surface charged residues. The extraction behavior of native cytochrome b(5) and modified proteins with substitutions of the type glutamic acid --> lysine at positions 44 (E44K), 56 (E56K), and 92 (E92K), was studied as a function of pH. The results indicate that an important mechanism of extraction is an electrostatic interaction of this protein with the negatively charged surfactant. We demonstrate that it is possible to improve extraction by engineering the protein surface charge, increasing the driving force responsible for the protein transfer to the micellar phase. (c) 1994 John Wiley & Sons, Inc.     

The structure of the S127P mutant of cytochrome b5 reductase that causes methemoglobinemia shows the AMP moiety of the flavin occupying the substrate binding site

Methemoglobinemia, the first hereditary disease to be identified that involved an enzyme deficiency, has been ascribed to mutations in the enzyme cytochrome b(5) reductase. A variety of defects in either the erythrocytic or microsomal forms of the enzyme have been identified that give rise to the type I or type II variant of the disease, respectively. The positions of the methemoglobinemia-causing mutations are scattered throughout the protein sequence, but the majority of the nontruncated mutants that produce type II symptoms occur close to the flavin adenine dinucleotide (FAD) cofactor binding site. While X-ray structures have been determined for the soluble, flavin-containing diaphorase domains of the rat and pig enzymes, no X-ray or NMR structure has been described for the human enzyme or any of the methemoglobinemia variants. S127P, a mutant that causes type II methemoglobinemia, was the first to be positively identified and have its spectroscopic and kinetic properties characterized that revealed altered nicotinamide adenine dinucleotide hydride (NADH) substrate binding behavior. To understand these changes at a structural level, we have determined the structure of the S127P mutant of rat cytochrome b(5) reductase to 1.8 A resolution, providing the first structural snapshot of a cytochrome b(5) reductase mutant that causes methemoglobinemia. The high-resolution structure revealed that the adenosine diphosphate (ADP) moiety of the FAD prosthetic group is displaced into the corresponding ADP binding site of the physiological substrate, NADH, thus acting as a substrate inhibitor which is consistent with both the spectroscopic and kinetic data.     

Kinetic properties of purified sheep lung microsomal NADH-cytochrome b5 reductase.

1. Lung NADH-cytochrome b5 reductase was saturated with its artificial substrate, potassium ferricyanide at approximately 0.1 mM ferricyanide concentration, and the activity of the lung enzyme was inhibited by the higher concentrations of potassium ferricyanide. Ferricyanide at 0.5 and 1.0 mM inhibited the activity of the enzyme by about 20 and 61% respectively. The apparent Km value was calculated as 13.7 microM potassium ferricyanide and 4.3 microM NADH. 2. The Michaelis constants for cytochrome b5 and NADH were determined to be 1.67 and 7.7 microM from the Lineweaver-Burk plots. These results demonstrate that affinity of the lung reductase for its natural substrate is almost 10 times higher than that for potassium ferricyanide. 3. Addition of non-ionic detergent stimulated the rate of reductase-catalyzed reduction of lung cytochrome b5 up to 8.2-fold. 4. Kinetic studies performed with lung reductase by varying NADH and cytochrome b5 concentrations at different fixed concentrations at cytochrome b5 or NADH showed a series of parallel lines indicating a "ping-pong" type of kinetic mechanism for interaction of NADH and cytochrome b5 with lung cytochrome b5 reductase.     

Production of a recombinant hybrid hemoflavoprotein: engineering a functional NADH:cytochrome c reductase.

A gene has been constructed coding for a unique fusion protein, NADH:cytochrome c reductase, that comprises the soluble heme-containing domain of rat hepatic cytochrome b(5) as the amino-terminal portion of the protein and the soluble flavin-containing domain of rat hepatic cytochrome b(5) reductase as the carboxyl terminus. The gene has been expressed in Escherichia coli resulting in the highly efficient production of a functional hybrid hemoflavoprotein which has been purified to homogeneity by a combination of ammonium sulfate precipitation, affinity chromatography on 5'-ADP agarose, and size-exclusion chromatography. The purified protein exhibited a molecular mass of approximately 46 kDa by polyacrylamide gel electrophoresis and 40,875 Da, for the apoprotein, using mass spectrometry which also confirmed the presence of both heme and FAD prosthetic groups. The fusion protein showed immunological cross-reactivity with both anti-rat cytochrome b(5) and anti-rat cytochrome b(5) reductase antibodies indicating the conservation of antigenic determinants from both native domains. Spectroscopic analysis indicated the fusion protein contained both a b-type cytochrome and flavin chromophors with properties identical to those of the native proteins. Amino-terminal and internal amino acid sequencing confirmed the identity of peptides derived from both the heme- and flavin-binding domains with sequences identical to the deduced amino acid sequence. The isolated fusion protein retained NADH:ferricyanide reductase activity (k(cat) = 8.00 x 10(2) s(-1), K(NADH)(m) = 4 microM, K(FeCN(6))(m) = 11 microM) comparable to that of that of native NADH:cytochrome b(5) reductase and also exhibited both NADH:cytochrome c reductase activity (k(cat) = 2.17 x 10(2) s(-1), K(NADH)(m) = 2 microM, K(FeCN(6))(m) = 11 microM, K(Cyt.c)(m) = 1 microM) and NADH:methemoglobin reductase activity (k(cat) = 4.40 x 10(-1) s(-1), K(NADH)(m) = 3 microM, K(mHb)(m) = 47 microM), the latter two activities indicating efficient electron transfer from FAD to heme and retention of physiological function. This work represents the first successful bacterial expression of a soluble, catalytically competent, rat hepatic cytochrome b(5)-cytochrome b(5) reductase fusion protein that retains the functional properties characteristic of the individual heme and flavin domain.     

Proapoptotic activity of cytochrome c in living cells: effect of K72 substitutions and species differences

Cytochrome c is one of the key proteins involved in the programmed cell death, and lysine 72 is known to be required for its apoptogenic activity. We have engineered a number of horse and murine cytochrome c single-point mutants with various substitutions at position 72 and compared quantitatively their proapoptotic activity in living cells. Apoptosis was activated by transferring exogenous cytochrome c into the cytoplasm of cells via a nontraumatic electroporation procedure. All mutant proteins studied exhibited significantly reduced proapoptotic activities in comparison with those for the wild type cytochromes. Relative activity of the horse (h(K72X)) and murine (m(K72W)) mutant proteins diminished in the order: h(K72R) > h(K72G) > h(K72A) > h(K72E) > h(K72L) > h(K72W) > m(K72W). As estimated, the horse and murine K72W mutants were at least 200- and 500-fold less active than corresponding wild type proteins. Thus, the K72W-substituted cytochrome c can serve as an adequate candidate for knock-in studies of cytochrome c-mediated apoptosis. The proapoptotic activity of wild-type cytochrome c from different species in murine monocytic WEHI-3 cells reduced in the order: murine cytochrome c > human cytochrome c approximately horse cytochrome c, thus indicating that apoptotic effect of cytochrome c depends on the species compatibility.     

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

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

Oxidation-reduction properties of the cytochrome b found in the plasma-membrane fraction of human neutrophils. A possible oxidase in the respiratory burst.

The oxidation-reduction midpoint potential of the cytochrome b found in the plasma membrane of human neutrophils has been determined at pH 7.0 (Em,7.0) from measurements of absorption spectra at fixed potentials. In both unstimulated and phorbol myristate acetate-stimulated cells Em,7.0 was -245 mV. Changes in pH affected the Em of the cytochrome b, with a slope of approx. 25 mV/pH unit change. The Em,7.0 of the haem group(s) of the membrane-bound myeloperoxidase of human neutrophils was found to be +34 mV. The plasma membranes contained no detectable ubiquinone, and no iron-sulphur compounds were detected by e.p.r. spectroscopy at 5-20 K. No flavins were detected by e.p.r. spectroscopy. The cytochrome b-245 was not reduced by added NADH or NADPH. Dithionite-reduced cytochrome b-245 formed a complex with CO, supplied as a saturated solution, which was dissociated with 26 microseconds illumination from a xenon flash lamp, and the recombination with CO had a half-time of approx. 6 ms. Partly (80%) reduced cytochrome b-245 was oxidized by added air-saturated buffer with a half-time faster than 1 s at 20 degrees C, a resolution limited by mixing time. These results are compatible with cytochrome b-245 acting as an oxidase.     

Structure of a cDNA for Ciona Cytochrome b(5) and the ubiquitous expression of mRNA in embryonic tissues

A cDNA clone for cytochrome b(5) was isolated from a cDNA library of an ascidian, Ciona savignyi, by a plaque hybridization method using a digoxigenin-labeled cDNA for the soluble form of human cytochrome b(5). The cDNA is composed of 5'- and 3'-noncoding sequences, and a 396-base pair coding sequence. The 3'-noncoding sequence contains polyadenylation signal sequences. The amino acid sequence of 132 residues deduced from the nucleotide sequence of the cDNA showed 61% identity and 82% similarity to the cytochrome b(5) of another ascidian species, Polyandrocarpa misakiensis, which we previously cloned. The amino-terminal hydrophilic domain of 98 residues contains well-conserved structures around two histidine residues for heme binding. A cDNA expression system was constructed to prepare a putative soluble form of Ciona cytochrome b(5). The recombinant soluble cytochrome b(5) showed an asymmetrical absorption spectrum at 560 nm as is shown by mammalian cytochromes b(5) upon reduction with NADH and NADH-cytochrome b(5) reductase. The recombinant Ciona cytochrome b(5) is reduced by NADH-cytochrome b(5) reductase with an apparent K(m) value of 3.3 microM. This value is similar to that of the cytochrome b(5) of Polyandrocarpa misakiensis. The expression of Ciona cytochrome b(5) mRNA during development was examined by an in situ hybridization method and ubiquitous expression in embryonic tissues was observed. The results indicate that cytochrome b(5) plays important roles in various metabolic processes during development.     

Structure and properties of the recombinant NADH-cytochrome b5 reductase of Physarum polycephalum

A cDNA for NADH-cytochrome b(5) reductase of Physarum polycephalum was cloned from a cDNA library, and the nucleotide sequence of the cDNA was determined (accession no. AB259870). The DNA of 943 base pairs contains 5'- and 3'-noncoding sequences, including a polyadenylation sequence, and a coding sequence of 843 base pairs. The amino acid sequence (281 residues) deduced from the nucleotide sequence was 25 residues shorter than those of vertebrate enzymes. Nevertheless, the recombinant Physarum enzyme showed enzyme activity comparable to that of the human enzyme. The recombinant Physarum enzyme showed a pH optimum of around 6.0, and apparent K(m) values of 2 microM and 14 microM for NADH and cytochrome b(5) respectively. The purified recombinant enzyme showed a typical FAD-derived absorption peak of cytochrome b(5) reductase at around 460 nm, with a shoulder at 480 nm. These results suggest that the Physarum enzyme plays an important role in the organism.     

Soluble NADH-cytochrome b5 reductase from rabbit liver cytosol: partial purification and characterization.

A soluble form of NADH-cytochrome b5 reductase (NADH: ferricytochrome b5 oxidoreductase, EC 1.6.2.2) was found in the cytosolic fraction of rabbit liver. The partially purified enzyme was strictly specific for NADH. It catalyzed the reduction of several substrates such as the methemoglobin-ferrocyanide complex (Hegesh, E. and Avron, M. (1967) Biochim. Biophys. Acta 146, 91-101) (apparent Km: 8 micrometer), potassium ferricyanide (apparent Km: 10 micrometer) and ferricytochrome b5 (apparent Km: 15 micrometer). Upon acrylamide gel isoelectro-focusing followed by specific staining, the enzyme was resolved into four bands (isoelectric pH: 7.05, 6.70, 6.50 and 6.30). The optimum pH of activity with ferricytochrome b5 as a substrate was 6.5. The estimated molecular weight was 25 000--30 000. The enzyme was unsensitive to cyanide. It was strongly inhibited by p-hydroxymercuribenzoate. The cytosolic liver cytochrome b5 reductase was immunologically related to the soluble cytochrome b5 reductase from human and rabbit red-cells, and to the microsomal cytochrome b5 reductase from rabbit liver.