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).     

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.     

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.     

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.     

Spinach Nitrate Reductase : Effects of Ionic Strength and pH on the Full and Partial Enzyme Activities

Initial velocity studies of immunopurified spinach nitrate reductase have been performed under conditions of controlled ionic strength and pH and in the absence of chloride ions. Increased ionic strength stimulated NADH:ferricyanide reductase and reduced flavin:nitrate reductase activities and inhibited NADH:nitrate reductase, NADH:cytochrome c reductase and reduced methyl viologen:nitrate reductase activities. NADH:dichlorophenolindophenol reductase activity was unaffected by changes in ionic strength. All of the partial activities, expressed in terms of micromole 2 electron transferred per minute per nanomole heme, were faster than the overall full, NADH:nitrate reductase activity indicating that none of the partial activities included the rate limiting step in electron transfer from NADH to nitrate. The pH optimum for NADH:nitrate reductase activity was determined to be 7 while values for the various partial activities ranged from 6.5 to 7.5. Chlorate, bromate, and iodate were determined to be alternate electron acceptors for the reduced enzyme. These results indicate that unlike the enzyme from Chlorella vulgaris, intramolecular electron transfer between reduced heme and Mo is not rate limiting for spinach nitrate reductase.     

High-level expression in Escherichia coli of the catalytically active flavin domain of corn leaf NADH:nitrate reductase and its comparison to human NADH:cytochrome B5 reductase

Higher plant nitrate reductase can be divided into three functional domains representing its prosthetic groups: 1) flavin; 2) cytochrome b; and 3) Mo-pterin. The flavin domain has been synthesized by heterologous expression in Escherichia coli using a fragment of a corn leaf NADH:nitrate reductase cDNA clone, Zmnr1, which we had previously isolated and sequenced. A Xho2-BamH1 fragment was cut from Zmnr1, containing the sequence for the flavin domain, and ligated in the BamH1 site of expression vector pET3c. When this construct was expressed in E. coli, a 30 kD polypeptide was found to be newly synthesized. The flavin domain was purified to homogeneity using blue Sepharose and shown to have a molecular weight of 30 kD. The recombinant flavin domain has a ferricyanide reductase specific activity of 1000 mumols NADH oxidized/min/mg protein and a visible spectrum virtually identical to that of human NADH:cytochrome b5 reductase.     

The circadian rhythm in Bryophyllum leaves: Phase control by radiant energy

An inactivated nitrate reductase (EC 1.6.6.1) formed in vivo by the green alga Chlorella fusca Shihira and Kraus is shown to be a cyanide complex. The partially purified inactive enzyme releases 0.048 nmol of HCN per unit of enzyme activated. This compares with 0.066 nmol of HCN liberated in similar previous measurements with the inactivated enzyme from Chlorella vulgaris. The nitrate reductase from C. fusca has been purified to a level of 67 mol nitrate reduced per min per mg enzyme. It contains a cytochrome b₅₅₇, at a level 1.9-fold higher per unit of active enzyme, than the nitrate reductase from C. vulgaris.Abbreviations FAD flavin-adenine dinucleotide - NADH nicotineamide-adenine-dinucleotide (reduced)     

Limited proteolysis of the nitrate reductase from spinach leaves

The functional structure of assimilatory NADH-nitrate reductase from spinach leaves was studied by limited proteolysis experiments. After incubation of purified nitrate reductase with trypsin, two stable products of 59 and 45 kDa were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fragment of 45 kDa was purified by Blue Sepharose chromatography. NADH-ferricyanide reductase and NADH-cytochrome c reductase activities were associated with this 45-kDa fragment which contains FAD, heme, and NADH binding fragment. After incubation of purified nitrate reductase with Staphylococcus aureus V8 protease, two major peaks were observed by high performance liquid chromatography size exclusion gel filtration. FMNH2-nitrate reductase and reduced methyl viologen-nitrate reductase activities were associated with the first peak of 170 kDa which consists of two noncovalently associated (75-90-kDa) fragments. NADH-ferricyanide reductase activity, however, was associated with the second peak which consisted of FAD and NADH binding sites. Incubation of the 45-kDa fragment with S. aureus V8 protease produced two major fragments of 28 and 14 kDa which contained FAD and heme, respectively. These results indicate that the molybdenum, heme, and FAD components of spinach nitrate reductase are contained in distinct domains which are covalently linked by exposed hinge regions. The molybdenum domain appears to be important in the maintenance of subunit interactions in the enzyme complex.     

Structure of a novel c7-type three-heme cytochrome domain from a multidomain cytochrome c polymer

The structure of a novel c(7)-type cytochrome domain that has two bishistidine coordinated hemes and one heme with histidine, methionine coordination (where the sixth ligand is a methionine residue) was determined at 1.7 A resolution. This domain is a representative of domains that form three polymers encoded by the Geobacter sulfurreducens genome. Two of these polymers consist of four and one protein of nine c(7)-type domains with a total of 12 and 27 hemes, respectively. Four individual domains (termed A, B, C, and D) from one such multiheme cytochrome c (ORF03300) were cloned and expressed in Escherichia coli. The domain C produced diffraction quality crystals from 2.4 M sodium malonate (pH 7). The structure was solved by MAD method and refined to an R-factor of 19.5% and R-free of 21.8%. Unlike the two c(7) molecules with known structures, one from G. sulfurreducens (PpcA) and one from Desulfuromonas acetoxidans where all three hemes are bishistidine coordinated, this domain contains a heme which is coordinated by a methionine and a histidine residue. As a result, the corresponding heme could have a higher potential than the other two hemes. The apparent midpoint reduction potential, E(app), of domain C is -105 mV, 50 mV higher than that of PpcA.     

The type I/type II cytochrome c3 complex: an electron transfer link in the hydrogen-sulfate reduction pathway

In Desulfovibrio metabolism, periplasmic hydrogen oxidation is coupled to cytoplasmic sulfate reduction via transmembrane electron transfer complexes. Type II tetraheme cytochrome c3 (TpII-c3), nine-heme cytochrome c (9HcA) and 16-heme cytochrome c (HmcA) are periplasmic proteins associated to these membrane-bound redox complexes and exhibit analogous physiological function. Type I tetraheme cytochrome c3 (TpI-c3) is thought to act as a mediator for electron transfer from hydrogenase to these multihemic cytochromes. In the present work we have investigated Desulfovibrio africanus (Da) and Desulfovibrio vulgaris Hildenborough (DvH) TpI-c3/TpII-c3 complexes. Comparative kinetic experiments of Da TpI-c3 and TpII-c3 using electrochemistry confirm that TpI-c3 is much more efficient than TpII-c3 as an electron acceptor from hydrogenase (second order rate constant k = 9 x 10(8) M(-1) s(-1), K(m) = 0.5 microM as compared to k = 1.7 x 10(7) M(-1) s(-1), K(m) = 40 microM, for TpI-c3 and TpII-c3, respectively). The Da TpI-c3/TpII-c3 complex was characterized at low ionic strength by gel filtration, analytical ultracentrifugation and cross-linking experiments. The thermodynamic parameters were determined by isothermal calorimetry titrations. The formation of the complex is mainly driven by a positive entropy change (deltaS = 137(+/-7) J mol(-1) K(-1) and deltaH = 5.1(+/-1.3) kJ mol(-1)) and the value for the association constant is found to be (2.2(+/-0.5)) x 10(6) M(-1) at pH 5.5. Our thermodynamic results reveal that the net increase in enthalpy and entropy is dominantly produced by proton release in combination with water molecule exclusion. Electrostatic forces play an important role in stabilizing the complex between the two proteins, since no complex formation is detected at high ionic strength. The crystal structure of Da TpI-c3 has been solved at 1.5 angstroms resolution and structural models of the complex have been obtained by NMR and docking experiments. Similar experiments have been carried out on the DvH TpI-c3/TpII-c3 complex. In both complexes, heme IV of TpI-c3 faces heme I of TpII-c3 involving basic residues of TpI-c3 and acidic residues of TpII-c3. A secondary interacting site has been observed in the two complexes, involving heme II of Da TpII-c3 and heme III of DvH TpI-c3 giving rise to a TpI-c3/TpII-c3 molar ratio of 2:1 and 1:2 for Da and DvH complexes, respectively. The physiological significance of these alternative sites in multiheme cytochromes c is discussed.     

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.     

The sequence of squash NADH:nitrate reductase and its relationship to the sequences of other flavoprotein oxidoreductases. A family of flavoprotein pyridine nucleotide cytochrome reductases.

Nucleotide sequences were determined for cDNA clones for squash NADH:nitrate oxidoreductase (EC 1.6.6.1), which is one of the most completely characterized forms of this higher plant enzyme. An open reading frame of 2754 nucleotides began at the first ATG. The deduced amino acid sequence contains 918 residues, with a predicted Mr = 103,376. The amino acid sequence is very similar to sequences deduced for other higher plant nitrate reductases. The squash sequence has significant similarity to the amino acid sequences of sulfite oxidase, cytochrome b5, and NADH:cytochrome b5 reductase. Alignment of these sequences with that of squash defines domains of nitrate reductase that appear to bind its 3 prosthetic groups (molybdopterin, heme-iron, and FAD). The amino acid sequence of the FAD domain of squash nitrate reductase was aligned with FAD domain sequences of other NADH:nitrate reductases, NADH:cytochrome b5 reductases, NADPH:nitrate reductases, ferredoxin:NADP+ reductases, NADPH:cytochrome P-450 reductases, NADPH:sulfite reductase flavoproteins, and Bacillus megaterium cytochrome P-450BM-3. In this multiple alignment, 14 amino acid residues are invariant, which suggests these proteins are members of a family of flavoenzymes. Secondary structure elements of the structural model of spinach ferredoxin:NADP+ reductase were used to predict the secondary structure of squash nitrate reductase and the other related flavoenzymes in this family. We suggest that this family of flavoenzymes, nearly all of which reduce a hemoprotein, be called "flavoprotein pyridine nucleotide cytochrome reductases."     

Regulation of Chlorella nitrate reductase: control of enzyme activity and immunoreactive protein levels by ammonia

Nitrate reductase catalyzes the initial step in the conversion of nitrate to organic nitrogen and is thought to be repressed by ammonia and induced by nitrate. Induction by nitrate and repression by ammonia were studied by following changes in NADH:nitrate reductase and the associated partial activities NADH:cytochrome c reductase and methylviologenr:nitrate reductase. Immunoreactive protein was assessed by enzyme-linked immunosorbent assay and immunoblotting. Molybdenum cofactor levels were investigated using the nit-1 complementation assay as well as fluorescence of the oxidized cofactor. The results indicate that the NADH:cytochrome c reductase activity is "induced" faster than the nitrate-reducing activity and suggest that incorporation of the molybdo-pterin cofactor may be rate limiting in the expression of activity. Molybdenum cofactor levels are significantly elevated in nitrate-treated cells. Under "repressing" conditions all activities decreased at approximately the same rate. A more rapid conversion of the enzyme to a reversibly inactive form also occurred under these conditions. Changes in immunoreactive protein levels correlated most closely with NADH:cytochrome c reductase activity but appeared to increase faster during induction and decrease slightly slower during repression than the enzyme activities. Removal of exogenous ammonia results in the appearance of nitrate reducing activity, as well as immunoreactive protein (derepression). Studies using protein and RNA synthesis inhibitors indicated that de novo synthesis is required for nitrate reductase induction and were in agreement with the results of the immunoreactive studies.     

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.     

Electrochemical and kinetic analysis of electron-transfer reactions of Chlorella nitrate reductase.

Assimilatory nitrate reductase (NR) from Chlorella is homotetrameric, each subunit containing FAD, heme, and Mo-pterin in a 1:1:1 stoichiometry. Measurements of NR activity and steady-state reduction of the heme component under conditions of NADH limitation or competitive inhibition by nitrite suggested intramolecular electron transfer between heme and Mo-pterin was a rate-limiting step and provided evidence that heme is an obligate intermediate in the transfer of electrons between FAD and Mo-pterin. In addition to the physiological substrates NADH and nitrate, various redox mediators undergo reactions with one or more of the prosthetic groups. These reactions are coupled by NR to NADH oxidation or nitrate reduction. To test whether intramolecular redox reactions of NR were rate-determining, rate constants for redox reactions between NR and several chemically diverse mediators were measured by cyclic voltammetry in the presence of NADH or nitrate. Reduction of ferrocenecarboxylic acid, dichlorophenolindophenol, and cytochrome c by NADH-reduced NR was coupled to reoxidation at a glassy carbon electrode (ferrocene and dichlorophenolindophenol) or at a bis(4-pyridyl) disulfide modified gold electrode (cytochrome c), yielding rate constants of 10.5 x 10(6), 1.7 x 10(6), and 2.7 x 10(6) M-1 s-1, respectively, at pH 7. Kinetics were consistent with a second-order reaction, implying that intramolecular heme reduction by NADH and endogenous FAD was not limiting. In contrast, reduction of methyl viologen and diquat at a glassy carbon electrode, coupled to oxidation by NR and nitrate, yielded similar kinetics for the two dyes. In both cases, second-order kinetics were not obeyed, and reoxidation of dye-reduced Mo-pterin of NR by nitrate became limiting at low scan rates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of NADH: nitrate reductase from the coccolithophorid Emiliania huxleyi (Lohman) Hay & Mohler (Haptophyceae)

Abstract Nitrate reductase was purified from and characterized in a bloom-forming unicellular calcifying alga, Emiliania huxleyi (Haptophyceae). The molecular masses of the native form and the subunit were 514 and 85 kDa, respectively, showing that the enzyme is a hexamer composed of 6 homologous subunits. The K _m values for NADH and NO3− were 40 μM and 104 μM, respectively. Activity of the reduction of nitrate was very high with reduced methylviologen and NADH, but no activity was observed with NADPH or reduced flavin mononucleotide; oxidation of NADH was very high with cytochrome c but did not occur with ferricyanide. These results indicate that Emiliania nitrate reductase is NADH-specific (EC 1.6.6.1), and that among algae and plants its subunit structure and kinetic properties are unique.     

Assimilatory nitrate reductase from Chlorella. Effect of ionic strength and pH on catalytic activity

Initial velocity studies of Chlorella nitrate reductase showed that increased ionic strength stimulated NADH:nitrate reductase activity by increasing both Vmax and Km for nitrate. Examination of the effect of ionic strength on the various partial activities of nitrate reductase revealed that while NADH:ferricyanide and reduced methyl viologen:nitrate reductase activities were unaffected by ionic strength, NADH:cytochrome c and reduced flavin:nitrate reductase activities were inhibited and stimulated by increased ionic strength, respectively. Comparison of the rates for the partial activities indicated electron transfer from heme to molybdenum to be the rate-limiting step in enzyme turnover. The pH optimum for NADH:nitrate reductase activity was found to be 7.9 while values for the partial activities ranged from 5.5 to 8.1. Phosphate was found to stimulate both NADH:nitrate and reduced methyl viologen:nitrate reductase activities indicating the molybdenum center as the site of interaction.     

Nitrate reductase of Neurospora crassa: the functional role of individual amino acids in the heme domain as examined by site-directed mutagenesis

The enzyme nitrate reductase, which catalyzes the reduction of nitrate to nitrite, is a multi-redox center homodimeric protein. Each polypeptide subunit is approximately 100 kDa in size and contains three separate domains, one each for a flavin, a heme-iron, and a molybdopterin cofactor. The heme-iron domain of nitrate reductase has homology with the simple redox protein, cytochrome b5, whose crystal structure was used to predict a three-dimensional structure for the heme domain. Two histidine residues have been identified that appear to coordinate the iron of the heme moiety, while other residues may be important in the folding or the function of the heme pocket. Site-directed mutagenesis was employed to obtain mutants that encode nitrate reductase derivatives with eight different single amino acid substitutions within the heme domain, including the two central histidine residues. Replacement of one of these histidines by alanine resulted in a completely nonfunctional enzyme whereas replacement of the other histidine resulted in a stable and functional enzyme with a lower affinity for heme. Certain amino acid substitutions appeared to cause a rapid turnover of the heme domain, whereas other substitutions were tolerated and yielded a stable and fully active enzyme. Three different single amino acid replacements within the heme domain led to a dramatic change in regulation of nitrate reductase synthesis, with significant expression of the enzyme even in the absence of nitrate induction.     

Cytochrome aa 3 from Methylococcus capsulatus (Bath)

A cytochrome aa ₃-type oxidase was isolated with and without a c-type cytochrome (cytochrome c-557) from Methylococcus capsulatus Bath by ion-exchange and hydrophobic chromatography in the presence of Triton X-100. Although cytochrome c-557 was not a constitutive component of the terminal oxidase, the cytochrome c ascorbate-TMPD oxidase activity of the enzyme decreased dramatically when the ratio of cytochrome c-557 to heme a dropped below 1:3. On denaturing gels, the purified enzyme dissociated into three subunits with molecular weights of 46,000, 28,000 and 20,000. The enzyme contains two heme groups (a and a ₃), absorption maximum at 422 nm in the resting state, at 445 and 601 nm in the dithionite reduced form and at 434 and 598 nm in the dithionite reduced plus CO form. Denaturing gels of the cytochrome aa ₃-cytochrome c-557 complex showed the polypeptides associated with cytochrome aa ₃ plus a heme c-staining subunit with a molecular weight of 37,000. The complex contains approximately two heme a, one heme c, absorption maximum at 420 nm in the resting state and at 421, 445, 522, 557 and 601 nm in the dithionite reduced form. The specific activity of the purified enzyme was 130 mol O₂/min · mol heme a compared to 753 mol O₂/min · mol heme a when isolated with cytochrome c-557.Abbreviations MMO methan monooxygenase - sMMO soluble methane monooxygenase - pMMO particulate methane monooxygenase - TMPD N,N,N,N-tetramethyl-p-phenylenediamine dihydrochloride - Na₂EDTA disodium ethylenediamine-tetraacetic acid     

Inhibition of the nitrate reductase complex from spinach by oxylamines

The nitrate reductase complex from spinach (Spinacia oleracea) was found to be inhibited by oxylamine compounds such as aminooxyacetate, hydroxylamine and O-methoxylamine. These compounds appear to interact with reduced cytochrome b₅₅₇ during catalysis of the enzyme. However, if the enzyme is maintained in a reduced state by NADH in the absence of nitrate, an additional component involved in FMNH₂-nitrate reductase is also affected by them. The binding of the oxylamines with the enzyme is non-covalent in nature as the inhibition can be reversed by treatment with 2-oxoglutarate.