Identification of quinone-binding and heme-ligating residues of the smallest membrane-anchoring subunit (QPs3) of bovine heart mitochondrial succinate:ubiquinone reductase.
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The smallest membrane-anchoring subunit (QPs3) of bovine heart succinate:ubiquinone reductase was overexpressed in Escherichia coli JM109 as a glutathione S-transferase fusion protein using the expression vector pGEX2T/QPs3. The yield of soluble active recombinant glutathione S-transferase-QPs3 fusion protein was isopropyl-1-thio-beta-D-galactopyranoside concentration-, induction growth time-, temperature-, and medium-dependent. Maximum yield of soluble recombinant fusion protein was obtained from cells harvested 3.5 h post-isopropyl-1-thio-beta-D-galactopyranoside (0.4 mM)-induction growth at 25 degrees C in 2.0% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 20 mM glucose (SOC medium) containing 440 mM sorbitol and 2.5 mM betaine. QPs3 was released from the fusion protein by proteolytic cleavage with thrombin. Isolated recombinant QPs3 shows one protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis that corresponds to subunit V of mitochondrial succinate:ubiquinone reductase. Although purified recombinant QPs3 is dispersed in 0.01% dodecylmaltoside, it is in a highly aggregated form, with an apparent molecular mass of more than 1 million. The recombinant QPs3 binds ubiquinone, causing a spectral blue shift. Upon titration of the recombinant protein with ubiquinone, a saturation behavior is observed, suggesting that the binding is specific and that recombinant QPs3 may be in the functionally active state. Two amino acid residues, serine 33 and tyrosine 37, in the putative ubiquinone binding domain of QPs3 are involved in ubiquinone binding because the S33A- or Y37A-substituted recombinant QPs3s do not cause the spectral blue shift of ubiquinone. Although recombinant QPs3 contains little cytochrome b560 heme, the spectral characteristics of cytochrome b560 are reconstituted upon addition of hemin chloride. Reconstituted cytochrome b560 in recombinant QPs3 shows a EPR signal at g = 2.92. Histidine residues at positions 46 and 60 are responsible for heme ligation because the H46N- or H60N-substituted QPs3 fail to restore cytochrome b560 upon addition of hemin chloride. (+info)
Anoxic function for the Escherichia coli flavohaemoglobin (Hmp): reversible binding of nitric oxide and reduction to nitrous oxide.
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The flavohaemoglobin Hmp of Escherichia coli is inducible by nitric oxide (NO) and provides protection both aerobically and anaerobically from inhibition of growth by NO and agents that cause nitrosative stress. Here we report rapid kinetic studies of NO binding to Fe(III) Hmp with a second order rate constant of 7.5 x 10(5) M(-1) s(-1) to generate a nitrosyl adduct that was stable anoxically but decayed in the presence of air to reform the Fe(III) protein. NO displaced CO bound to dithionite-reduced Hmp but, remarkably, CO recombined after only 2 s at room temperature indicative of NO reduction and dissociation from the haem. Addition of NO to anoxic NADH-reduced Hmp also generated a nitrosyl species which persisted while NADH was oxidised. These results are consistent with direct demonstration by membrane-inlet mass spectrometry of NO consumption and nitrous oxide production during anoxic incubation of NADH-reduced Hmp. The results demonstrate a new mechanism by which Hmp may eliminate NO under anoxic growth conditions. (+info)
Classes of Anabaena variabilis mutants with oxygen-sensitive nitrogenase activity.
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Mutants of Anabaena variabilis deficient in the envelope glycolipids of heterocysts have no or very low nitrogenase activity when assayed aerobically. Revertants capable of aerobic growth on N2 have increased quantities of these glycolipids. Among mutants which require fixed nitrogen for growth in air and which have a normal complement of glycolipids, one expresses high nitrogenase activity at low oxygen tension. Three others show high nitrogenase activity only in the presence of dithionite and are therefore impaired in electron transfer. (+info)
Characterization of DorC from Rhodobacter capsulatus, a c-type cytochrome involved in electron transfer to dimethyl sulfoxide reductase.
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The dorC gene of the dimethyl sulfoxide respiratory (dor) operon of Rhodobacter capsulatus encodes a pentaheme c-type cytochrome that is involved in electron transfer from ubiquinol to periplasmic dimethyl sulfoxide reductase. DorC was expressed as a C-terminal fusion to an 8-amino acid FLAG epitope and was purified from detergent-solubilized membranes by ion exchange chromatography and immunoaffinity chromatography. The DorC protein had a subunit Mr = 46,000, and pyridine hemochrome analysis indicated that it contained 5 mol heme c/mol DorC polypeptide, as predicted from the derived amino acid sequence of the dorC gene. The reduced form of DorC exhibited visible absorption maxima at 551.5 nm (alpha-band), 522 nm (beta-band), and 419 nm (Soret band). Redox potentiometry of the heme centers of DorC identified five components (n = 1) with midpoint potentials of -34, -128, -184, -185, and -276 mV. Despite the low redox potentials of the heme centers, DorC was reduced by duroquinol and was oxidized by dimethyl sulfoxide reductase. (+info)
Nitric oxide inhibits L-type Ca2+ current in glomus cells of the rabbit carotid body via a cGMP-independent mechanism.
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Previous studies have shown that nitric oxide (NO) inhibits carotid body sensory activity. To begin to understand the cellular mechanisms associated with the actions of NO in the carotid body, we monitored the effects of NO donors on the macroscopic Ca2+ current in glomus cells isolated from rabbit carotid bodies. Experiments were performed on freshly dissociated glomus cells from adult rabbit carotid bodies using the whole cell configuration of the patch-clamp technique. The NO donors sodium nitroprusside (SNP; 600 microM, n = 7) and spermine nitric oxide (SNO; 100 microM, n = 7) inhibited the Ca2+ current in glomus cells in a voltage-independent manner. These effects of NO donors were rapid in onset and peaked within 1 or 2 min. In contrast, the outward K+ current was unaffected by SNP (600 microM, n = 6), indicating that the inhibition by SNP was not a nonspecific membrane effect. 2-(4-carboxyphenyl)-4,4,5, 5-tetramethyl-imidazoline-1-oxyl-3-oxide (carboxy-PTIO; 500 microM), an NO scavenger, prevented inhibition of the Ca2+ current by SNP (n = 7), whereas neither superoxide dismutase (SOD; 2,000 U/ml, n = 4), a superoxide scavenger, nor sodium hydrosulfite (SHS; 1 mM, n = 7), a reducing agent, prevented inhibition of the Ca2+ current by SNP. However, SNP inhibition of the Ca2+ current was reversible in the presence of either SOD or SHS. These results suggest that NO itself inhibits Ca2+ current in a reversible manner and that subsequent formation of peroxynitrites results in irreversible inhibition. SNP inhibition of the Ca2+ current was not affected by 30 microM LY 83, 583 (n = 7) nor was it mimicked by 600 microM 8-bromoguanosine 3':5'-cyclic monophosphate (8-Br-cGMP; n = 6), suggesting that the effects of NO on the Ca2+ current are mediated, in part, via a cGMP-independent mechanism. N-ethylmaleimide (NEM; 2.5 mM, n = 6) prevented the inhibition of the Ca2+ current by SNP, indicating that SNP is acting via a modification of sulfhydryl groups on Ca2+ channel proteins. Norepinephrine (NE; 10 microM) further inhibited the Ca2+ current in the presence of NEM (n = 7), implying that NEM did not nonspecifically eliminate Ca2+ current modulation. Nisoldipine, an L-type Ca2+ channel blocker (2 microM, n = 6), prevented the inhibition of Ca2+ current by SNP, whereas omega-conotoxin GVIA, an N-type Ca2+ channel blocker (1 microM, n = 9), did not prevent the inhibition of Ca2+ current by SNP. These results demonstrate that NO inhibits L-type Ca2+ channels in adult rabbit glomus cells, in part, due to a modification of calcium channel proteins. The inhibition might provide one plausible mechanism for efferent inhibition of carotid body activity by NO. (+info)
MgATP-independent hydrogen evolution catalysed by nitrogenase: an explanation for the missing electron(s) in the MgADP-AlF4 transition-state complex.
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When the MoFe (Kp1) and Fe (Kp2) component proteins of Klebsiella pneumoniae nitrogenase are incubated with MgADP and AlF4(-) in the presence of dithionite as a reducing agent, a stable putative transition-state complex is produced [Yousafzai and Eady (1997) Biochem. J. 326, 637-640]. Surprisingly, the EPR signal associated with reduced Kp2 is not detectable, but Kp1 retains the S=3/2 EPR signal arising from the dithionite reduced state of the MoFe cofactor centre of the protein. This is consistent with the [Fe4S4] centre of the Fe protein in the complex being oxidized, and similar observations have been made with the complex of Azotobacter vinelandii [Spee, Arendsen, Wassink, Marritt, Hagen and Haaker (1998) FEBS Lett. 432, 55-58]. No satisfactory explanation for the fate of the electrons lost by Kp2 has been forthcoming. However, we report here that during the preparation of the MgADP-AlF4 K. pneumoniae complex under argon, H2 was evolved in amounts corresponding to one half of the FeMoco content of the Kp1 (FeMoco is the likely catalytic site of nitrogenase with a composition Mo:Fe7:S9:homocitrate). This is surprising, since activity is observed during incubation in the absence of MgATP, normally regarded as being essential for nitrogenase function, and in the presence of MgADP, a strong competitive inhibitor of nitrogenase. The formation of H2 by nitrogenase in the absence of AlF4(-) was also observed in reaction mixtures containing MgADP but not MgATP. The reaction showed saturation kinetics when Kp1 was titrated with increasing amounts of Kp2 and, at saturation, the amount of H2 formed was stoichiometric with the FeMoco content of Kp1. The dependence of the rate of formation of H2 on [MgADP] was inconsistent with the activity arising from MgATP contamination. We conclude that MgATP is not obligatory for H+ reduction by nitrogenase since MgADP supports a very low rate of hydrogen evolution. (+info)
31P nuclear magnetic resonance study of the flavoprotein component of the Escherichia coli sulfite reductase.
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SiR-FP60, the monomeric form of the Escherichia coli sulfite reductase flavoprotein component (SiR-FP), has been analysed by 31P-NMR spectroscopy. This protein was reported previously as a reliable simplified model for native SiR-FP [Zeghouf, M., Fontecave, M., Macherel, D., & Coves, J. (1998) Biochemistry 37, 6117-6123]. SiR-FP60 was examined in its native form, as a complex with NADP+ and after monoelectronic reduction either with NADPH or dithionite. In these latter cases, the stabilized FMN semiquinone radical offers a natural and internal paramagnetic probe. The paramagnetic effect of added manganese was also studied. In each case, the NMR parameters were extracted from digitalized data by a deconvolution procedure and compared with those obtained previously with cytochrome P450 reductase. Evolution of the NMR parameters and of calculated relaxation rate constants upon biochemical modifications of SiR-FP60 led us to propose that the reactive center is more compact than the one of cytochrome P450 reductase, with the redox components, FMN, FAD and NADPH, in a tighter spatial arrangement, close to the protein surface. This underlies some subtle differences between the two proteins for which a very similar overall structure is likely considering their common genetic origin and common operating cycle. (+info)
Purification and catalytic properties of Ech hydrogenase from Methanosarcina barkeri.
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Methanosarcina barkeri has recently been shown to produce a multisubunit membrane-bound [NiFe] hydrogenase designated Ech (Escherichia coli hydrogenase 3) hydrogenase. In the present study Ech hydrogenase was purified to apparent homogeneity in a high yield. The enzyme preparation obtained only contained the six polypeptides which had previously been shown to be encoded by the ech operon. The purified enzyme was found to contain 0.9 mol of Ni, 11.3 mol of nonheme-iron and 10.8 mol of acid-labile sulfur per mol of enzyme. Using the purified enzyme the kinetic parameters were determined. The enzyme catalyzed the H2 dependent reduction of a M. barkeri 2[4Fe-4S] ferredoxin with a specific activity of 50 U x mg protein-1 at pH 7.0 and exhibited an apparent Km for the ferredoxin of 1 microM. The enzyme also catalyzed hydrogen formation with the reduced ferredoxin as electron donor at a rate of 90 U x mg protein-1 at pH 7.0. The apparent Km for the reduced ferredoxin was 7.5 microM. Reduction or oxidation of the ferredoxin proceeded at similar rates as the reduction or oxidation of oxidized or reduced methylviologen, respectively. The apparent Km for H2 was 5 microM. The kinetic data strongly indicate that the ferredoxin is the physiological electron donor or acceptor of Ech hydrogenase. Ech hydrogenase amounts to about 3% of the total cell protein in acetate-grown, methanol-grown or H2/CO2-grown cells of M. barkeri, as calculated from quantitative Western blot experiments. The function of Ech hydrogenase is ascribed to ferredoxin-linked H2 production coupled to the oxidation of the carbonyl-group of acetyl-CoA to CO2 during growth on acetate, and to ferredoxin-linked H2 uptake coupled to the reduction of CO2 to the redox state of CO during growth on H2/CO2 or methanol. (+info)