Detecting network communities by propagating labels under constraints.

We investigate the recently proposed label-propagation algorithm (LPA) for identifying network communities. We reformulate the LPA as an equivalent optimization problem, giving an objective function whose maxima correspond to community solutions. By considering properties of the objective function, we identify conceptual and practical drawbacks of the label-propagation approach, most importantly the disparity between increasing the value of the objective function and improving the quality of communities found. To address the drawbacks, we modify the objective function in the optimization problem, producing a variety of algorithms that propagate labels subject to constraints; of particular interest is a variant that maximizes the modularity measure of community quality. Performance properties and implementation details of the proposed algorithms are discussed. Bipartite as well as unipartite networks are considered.

Modularity and community detection in bipartite networks.

The modularity of a network quantifies the extent, relative to a null model network, to which vertices cluster into community groups. We define a null model appropriate for bipartite networks, and use it to define a bipartite modularity. The bipartite modularity is presented in terms of a modularity matrix B; some key properties of the eigenspectrum of B are identified and used to describe an algorithm for identifying modules in bipartite networks. The algorithm is based on the idea that the modules in the two parts of the network are dependent, with each part mutually being used to induce the vertices for the other part into the modules. We apply the algorithm to real-world network data, showing that the algorithm successfully identifies the modular structure of bipartite networks.

Multiple thresholds in a model system of noisy ion channels.

Voltage-activated ion channels vary randomly between open and closed states, influenced by the membrane potential and other factors. Signal transduction is enhanced by noise in a simple ion channel model. The enhancement occurs in a finite range of signals; the range can be extended using populations of channels. The range increases more rapidly in multiple-threshold channel populations than in single-threshold populations. The diversity of ion channels may thus be present as a strategy to reduce the metabolic costs of handling a broad class of electrochemical signals.

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.

Network of European Union-funded collaborative research and development projects.

We describe collaboration networks consisting of research projects funded by the European Union (EU) and the organizations involved in those projects. The networks are substantial in terms of size, complexity, and potential impact on research policies and national economies in the EU. In empirical determinations of the network properties, we observe characteristics similar to those of other collaboration networks, including scale-free degree distributions, small diameter, and high clustering. We present some plausible models for the formation and structure of networks with the observed properties.

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.

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.

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+.

Cytochrome b5 oxidoreductase: expression and characterization of the original familial ideopathic methemoglobinemia mutations E255- and G291D.

NADH:cytochrome b5 oxidoreductase catalyzes the transfer of reducing equivalents from the physiological electron donor, NADH, to two molecules of cytochrome b5. Utilizing a heterologous expression system for the soluble, catalytic domain of the rat microsomal enzyme, we have produced two mutants, corresponding to E255- and G291D. These mutants correspond to the two specific mutations that were identified over a half century later following diagnosis of the original cases of type I recessive congenital methemoglobinemia (RCM). We have purified both the E255- and G291D variants to homogeneity to determine the molecular basis for type I RCM in these individuals. Both the E255- and G291D variants retained a full complement of FAD and exhibited absorption and CD spectroscopic properties comparable to those of the wild-type protein. Oxidation-reduction potentiometric titrations yielded standard midpoint potentials (E0') for the FAD/FADH2 couple of -271 and -273 mV for the E255- and G291D variants, respectively, which were comparable to the value of -268 mV obtained for the wild-type protein and confirmed that the redox potential of the flavin was unaffected by either mutation. Thermal and proteolytic stability studies revealed that while the G291D variant exhibited stability comparable to that of wild-type, the E255- variant was markedly less stable, indicative of an altered conformation. Initial-rate kinetic studies revealed that both mutants had decreased catalytic activity (kcat), with the E255- and G291D variants retaining approximately 38 and 58% of wild-type activity, respectively. However, the affinity for NADH (KmNADH) was decreased approximately 100-fold for E255- compared to only approximately 1.3-fold for G291D, results supported by the spectroscopic binding constant (Ks) obtained for G291D. These results indicate that the properties of both the E255- and G291D cytochrome b5 oxidoreductase mutants are similar to those of other variants that have been identified as resulting in the type I form of RCM.

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.

Voltammetric simulations of multiple electron transfer/proton transfer coupled reactions: flavin adenine dinucleotide as a model system.

Cyclic voltammograms were simulated using DigiSim software for reaction mechanisms involving multiple electron transfer steps coupled to proton transfer. Specifically, the overall reaction mechanism of the form O+2e(-)+2H(+) right harpoon over left harpoon RH(2) was used to simulate experimental reduction potentials as a function of pH. The pH-dependent reduction potentials reported in the literature for flavodoxin and free flavin adenine dinucleotide were simulated based on selected reduction potentials and acid dissociation constants. Relationships between reduction potentials and acid dissociation constants are presented to model n=1 and n=2 reaction mechanisms and one- and two-proton-coupled redox reactions. Experimental parameters used in the simulations were selected such that the electron and proton transfer reactions were not rate limiting, and therefore these simulated reactions involve thermodynamic coupling rather than concerted kinetic processes.

Engineering and characterization of a NADPH-utilizing cytochrome b5 reductase.

Microsomal cytochrome b(5) reductase (EC 1.6.2.2) catalyzes the reduction of ferricytochrome b(5) using NADH as the physiological electron donor. Site-directed mutagenesis has been used to engineer the soluble rat cytochrome b(5) reductase diaphorase domain to utilize NADPH as the preferred electron donor. Single and double mutations at residues D239 and F251 were made in a recombinant expression system that corresponded to D239E, S and T, F251R, and Y, D239S/F251R, D239S/F251Y, and D239T/F251R, respectively. Steady-state turnover measurements indicated that D239S/F251Y was bispecific while D239T, D239S/F251R, and D239T/F251R were each NADPH-specific. Wild-type (WT) cytochrome b(5) reductase showed a 3700-fold preference for NADH whereas the mutant with the highest NADPH efficiency, D239T, showed an 11-fold preference for NADPH, a 39200-fold increase. Wild-type cytochrome b(5) reductase only formed a stable charge-transfer complex with NADH while D239T formed complexes with both NADH and NADPH. The rates of hydride ion transfer, determined by stopped-flow kinetics, were k(NADH-WT) = 130 s(-1), k(NADPH-WT) = 5 s(-1), k(NADH-D239T) = 180 s(-1), and k(NADPH-D239T) = 73 s(-1). K(s) determinations by differential spectroscopy demonstrated that D239T could bind nonreducing pyridine nucleotides with a phosphate or a hydroxyl substituent at the 2' position, whereas wild-type cytochrome b(5) reductase would only bind 2' hydroxylated molecules. Oxidation-reduction potentials (E degrees ', n = 2) for the flavin cofactor were WT = -268 mV, D239T = -272 mV, WT+NAD(+) = -190 mV, D239T+NAD(+) = -206 mV, WT+NADP(+) = -253 mV, and D239T+NADP(+) = -215 mV, which demonstrated the thermodynamic contribution of NADP(+) binding to D239T. The crystal structures of D239T and D239T in complex with NAD(+) indicated that the loss of the negative electrostatic surface that precluded 2' phosphate binding in the wild-type enzyme was primarily responsible for the observed improvement in the use of NADPH by the D239T mutant.

Heterologous expression of enzymopenic methemoglobinemia variants using a novel NADH:cytochrome c reductase fusion protein.

Hereditary enzymopenic methemoglobinemia is a rare disease that predominantly results from defects in either the erythrocytic (type I) or microsomal (type II) forms of the enzyme NADH:cytochrome b5 reductase (EC 1.6.2.2). All 25 currently identified type I and type II methemoglobinemia mutants have been expressed in Escherichia coli using a novel six histidine-tagged rat cytochrome b5/cytochrome b5 reductase fusion protein designated NADH:cytochrome c reductase (H6NCR). All 25 H6NCR variants were isolated and demonstrated to result in two groups of expression products. The first group of 16 mutants, which included the majority of the type I mutants, included K116Q, P131L, L139P, T183S, M193V, S194P, P211L, L215P, A245T, A245V, C270Y, E279K, V305R, V319M, M340-, and F365-, and yielded full-length fusion proteins that retained variable levels of NADH:cytochrome c reductase (NADH:CR) activity, ranging from approximately 2% (M340-) to 92% (K116Q) of that of the wild-type fusion protein. In contrast, the remaining nine mutants that represented the majority of the type II variants, comprised a second group that included Y109*, R124Q, Q143*, R150*, P162H, V172M, R226*, C270R, and R285*, and resulted in truncated H6NCR variants that retained the amino-terminal cytochrome b5 domain but were devoid of NADH:CR activity due to the absence of the cytochrome b5 reductase flavin domain. Kinetic analyses of the first group of full-length mutant fusion proteins indicated that values for both kcat and Km(NADH) were decreased and increased, respectively, indicating that the various mutations affected both substrate affinity and/or turnover. However, for the second group, the truncated products were the result of incomplete production of the carboxyl-terminal flavin-containing domain or instability of the expression products due to improper folding and/or lack of flavin incorporation.

Biotin sulfoxide reductase: Tryptophan 90 is required for efficient substrate utilization.

Rhodobacter sphaeroides f. sp. denitrificans biotin sulfoxide reductase (BSOR) catalyzes the reduction of d-biotin d-sulfoxide to biotin and contains the molybdopterin guanine dinucleotide (MGD) cofactor as its sole prosthetic group. Comparison of the primary sequences of BSOR and the closely related enzyme dimethyl sulfoxide reductase (DMSOR) indicated a number of conserved residues, including an active-site tryptophan residue (W90), which has been suggested to be involved in hydrogen bonding to the oxo group on the Mo(VI) center in BSOR. Site-directed mutagenesis has been used to replace tryptophan 90 in BSOR with phenylalanine, tyrosine, and alanine residues to examine the role of this residue in catalysis. All three BSOR mutant proteins were purified to homogeneity and contained MGD. The mutant proteins retained very limited activity toward the oxidizing substrates tested, with W90F retaining the most activity (3.4% of wild type). All three W90 mutant proteins exhibited greatly reduced k(cat) values compared to that of the wild-type enzyme, which was accompanied by little change in K(mapp). In addition, the mutant proteins had perturbed visible absorption and circular dichroism spectra suggesting different oxidation states of the Mo center. Purified samples of wild-type BSOR did not exhibit electron paramagnetic resonance (EPR) signals indicating a Mo(VI) center. After redox-cycling, partially reduced samples of wild-type BSOR revealed a proton-split S=1/2 Mo(V) resonance (g(1,2,3)=1.999, 1.981, 1.967; A(1,2,3)=1.40, 1.00, 1.05 mT) analogous to that observed in DMSOR. In contrast, EPR studies of the purified W90 mutant proteins revealed distinct S=1/2 Mo(V) resonances that were resistant to both oxidation and reduction, indicating that the Mo was trapped in the intermediate Mo(V) oxidation state. These results strongly suggest that W90 in BSOR plays a critical role in catalysis by serving as a hydrogen bond donor to the oxo group on the Mo(VI) center.

Bacterial expression of the molybdenum domain of assimilatory nitrate reductase: production of both the functional molybdenum-containing domain and the nonfunctional tungsten analog.

Assimilatory NADH:nitrate reductase (EC 1.6.6.1), a complex molybdenum-, cytochrome b(557)- and FAD-containing protein, catalyzes the regulated and rate-limiting step in the utilization of inorganic nitrogen by higher plants. To facilitate structure/function studies of the individual molybdenum center, we have developed bacterial expression systems for the heterologous production of the 541 residue amino-terminal, molybdenum center-containing domain of spinach nitrate reductase either as a six-histidine-tagged variant or as a glutathione-S-transferase-tagged fusion protein. Expression of the his-tagged molybdenum domain in Escherichia coli BL21(DE3) cells under anaerobic conditions yielded a 55-kDa domain with a specific activity of 1.5 micromol NO(3)(-) consumed/min/nmol enzyme and with a K(mapp)(NO(3)(-)) of 8 mciroM. In contrast, expression of the molybdenum domain as a GST-tagged fusion protein in E. coli TP1000(MobA(-) strain) cells under aerobic conditions yielded an 85-kDa fusion protein with a specific activity of 10.8 micromol NO(3)(-) consumed/min/nmol enzyme and with a K(mapp)(NO(3)(-)) of 12 microM. Fluorescence analysis indicated that both forms of the molybdenum domain contained the cofactor, MPT, although the MPT content was higher in the GST-fusion domain. Inductively coupled plasma mass spectrometric analysis of both the his-tagged and GST-fusion protein domain samples indicated Mo/protein ratios of 0.44 and 0.93, respectively, confirming a very high level of Mo incorporation in the GST-fusion protein. Expression of the GST-fusion protein in TP1000 cells in the presence of elevated tungsten concentrations resulted in an 85-kDa fusion protein that contained MPT but which was devoid of nitrate-reducing activity. Partial reduction of the molybdenum domain resulted in the generation of an axial Mo(V) EPR species with g values of 1.9952, 1.9693, and 1.9665, respectively, and exhibiting superhyperfine coupling to a single exchangeable proton, analogous to that previously observed for the native enzyme. In contrast, the tungsten-substituted MPT-containing domain yielded a W(V) EPR species with g values of 1.9560, 1.9474, and 1.9271, respectively, with unresolved superhyperfine interaction. NADH:nitrate reductase activity could be reconstituted using the GST-molybdenum domain fusion protein in the presence of the recombinant forms of the spinach nitrate reductase' flavin- and heme-containing domains.

Synthesis and bacterial expression of a gene encoding the heme domain of assimilatory nitrate reductase.

Assimilatory NADH:nitrate reductase (EC 1.6.6.1), a complex Mo-pterin-, cytochrome b(557)-, and FAD-containing protein, catalyzes the regulated and rate-limiting step in the utilization of inorganic nitrogen by higher plants. A codon-optimized gene has been synthesized for expression of the central cytochrome b(557)-containing fragment, corresponding to residues A542-E658, of spinach assimilatory nitrate reductase. While expression of the full-length synthetic gene in Escherichia coli did not result in significant heme domain production, expression of a Y647* truncated form resulted in substantial heme domain production as evidenced by the generation of "pink" cells. The histidine-tagged heme domain was purified to homogeneity using a combination of NTA-agarose and size-exclusion FPLC, resulting in a single protein band following SDS-PAGE analysis with a molecular mass of approximately 13 kDa. MALDI-TOF mass spectrometry yielded an m/z ratio of 12,435 and confirmed the presence of the heme prosthetic group (m/z=622) while cofactor analysis indicated a 1:1 heme to protein stoichiometry. The oxidized heme domain exhibited spectroscopic properties typical of a b-type cytochrome with a visible Soret maximum at 413 nm together with epr g-values of 2.98, 2.26, and 1.49, consistent with low-spin bis-histidyl coordination. Oxidation-reduction titrations of the heme domain indicated a standard midpoint potential (E(o)') of -118 mV. The isolated heme domain formed a 1:1 complex with cytochrome c with a K(A) of 7 microM (micro=0.007) and reconstituted NADH:cytochrome c reductase activity in the presence of a recombinant form of the spinach nitrate reductase flavin domain, yielding a k(cat) of 1.4 s(-1) and a K(m app) for cytochrome c of 9 microM. These results indicate the efficient expression of a recombinant form of the heme domain of spinach nitrate reductase that retained the spectroscopic and thermodynamic properties characteristic of the corresponding domain in the native spinach enzyme.

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).