Publications

• Cytochrome cd1, reductive activation and kinetic analysis of a multifunctional respiratory enzyme.

  Richter C.D., Allen J.W., Higham C.W., Koppenhofer A., Zajicek R.S., Watmough N.J., Ferguson S.J.

  Paracoccus pantotrophus cytochrome cd(1) is an enzyme of bacterial respiration, capable of using nitrite in vivo and also hydroxylamine and oxygen in vitro as electron acceptors. We present a comprehensive analysis of the steady state kinetic properties of the enzyme with each electron acceptor an ... >> More

  Paracoccus pantotrophus cytochrome cd(1) is an enzyme of bacterial respiration, capable of using nitrite in vivo and also hydroxylamine and oxygen in vitro as electron acceptors. We present a comprehensive analysis of the steady state kinetic properties of the enzyme with each electron acceptor and three electron donors, pseudoazurin and cytochrome c(550), both physiological, and the non-physiological horse heart cytochrome c. At pH 5.8, optimal for nitrite reduction, the enzyme has a turnover number up to 121 s(-1) per d(1) heme, significantly higher than previously observed for any cytochrome cd(1). Pre-activation of the enzyme via reduction is necessary to establish full catalytic competence with any of the electron donor proteins. There is no significant kinetic distinction between the alternative physiological electron donors in any respect, providing support for the concept of pseudospecificity, in which proteins with substantially different tertiary structures can transfer electrons to the same acceptor. A low level hydroxylamine disproportionase activity that may be an intrinsic property of cytochromes c is also reported. Important implications for the enzymology of P. pantotrophus cytochrome cd(1) are discussed and proposals are made about the mechanism of reduction of nitrite, based on new observations placed in the context of recent rapid reaction studies. << Less

  J Biol Chem 277:3093-3100(2002) [PubMed] [EuropePMC]

• Hydroxylamine reductase from Pseudomonas aeruginosa.

  WALKER G.C., NICHOLAS D.J.

  Biochim Biophys Acta 49:361-368(1961) [PubMed] [EuropePMC]

• Hydroxylamine assimilation by Rhodobacter capsulatus E1F1. requirement of the hcp gene (hybrid cluster protein) located in the nitrate assimilation nas gene region for hydroxylamine reduction.

  Cabello P., Pino C., Olmo-Mira M.F., Castillo F., Roldan M.D., Moreno-Vivian C.

  Rhodobacter capsulatus E1F1 grows phototrophically with nitrate as nitrogen source. Using primers designed for conserved motifs in bacterial assimilatory nitrate reductases, a 450-bp DNA was amplified by PCR and used for the screening of a genomic library. A cosmid carrying an insert with four Sal ... >> More

  Rhodobacter capsulatus E1F1 grows phototrophically with nitrate as nitrogen source. Using primers designed for conserved motifs in bacterial assimilatory nitrate reductases, a 450-bp DNA was amplified by PCR and used for the screening of a genomic library. A cosmid carrying an insert with four SalI fragments of 2.8, 4.1, 4.5, and 5.8 kb was isolated, and DNA sequencing revealed that it contains a nitrate assimilation (nas) gene region, including the hcp gene coding for a hybrid cluster protein (HCP). Expression of hcp is probably regulated by a nitrite-sensitive repressor encoded by the adjacent nsrR gene. A His(6)-HCP was overproduced in Escherichia coli and purified. HCP contained about 6 iron and 4 labile sulfide atoms per molecule, in agreement with the presence of both [2Fe-2S] and [4Fe-2S-2O] clusters, and showed hydroxylamine reductase activity, forming ammonia in vitro with methyl viologen as reductant. The apparent K(m) values for NH(2)OH and methyl viologen were 1 mM and 7 microM, respectively, at the pH and temperature optima (9.3 and 40 degrees C). The activity was oxygen-sensitive and was inhibited by sulfide and iron reagents. R. capsulatus E1F1 grew phototrophically, but not heterotrophically, with 1 mM NH(2)OH as nitrogen source, and up to 10 mM NH(2)OH was taken up by anaerobic resting cells. Ammonium was transiently accumulated in the media, and its assimilation was prevented by L-methionine-D,L-sulfoximine, a glutamine synthetase inhibitor. In addition, hydroxylamine- or nitrite-grown cells showed the higher hydroxylamine reductase activities. However, R. capsulatus B10S, a strain lacking the whole hcp-nas region, did not grow with 1 mM NH(2)OH. Also, E. coli cells overproducing HCP tolerate hydroxyl-amine better during anaerobic growth. These results suggest that HCP is involved in assimilation of NH(2)OH, a toxic product that could be formed during nitrate assimilation, probably in the nitrite reduction step. << Less

  J. Biol. Chem. 279:45485-45494(2004) [PubMed] [EuropePMC]

• Hydroxylamine reductase activity of the hybrid cluster protein from Escherichia coli.

  Wolfe M.T., Heo J., Garavelli J.S., Ludden P.W.

  The hybrid cluster protein (HCP; formerly termed the prismane protein) has been extensively studied due to its unique spectroscopic properties. Although the structural and spectroscopic characteristics are well defined, its enzymatic function, up to this point, has remained unidentified. While it ... >> More

  The hybrid cluster protein (HCP; formerly termed the prismane protein) has been extensively studied due to its unique spectroscopic properties. Although the structural and spectroscopic characteristics are well defined, its enzymatic function, up to this point, has remained unidentified. While it was proposed that HCP acts in some step of nitrogen metabolism, a specific role for this enzyme remained unknown. Recent studies of HCP purified from Escherichia coli have identified a novel hydroxylamine reductase activity. These data reveal the ability of HCP to reduce hydroxylamine in vitro to form NH(3) and H(2)O. Further biochemical analyses were completed in order to determine the effects of various electron donors, different pH levels, and the presence of CN(-) on in vitro hydroxylamine reduction. << Less

  J. Bacteriol. 184:5898-5902(2002) [PubMed] [EuropePMC]