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Instruments / Technical Area	Publication Details	Published
I24-Microfocus Macromolecular Crystallography	Resolution of Key Roles for the Distal Pocket Histidine in Cytochrome Colin Lockwood , Bénédicte Burlat , Myles R. Cheesman , Melanie Kern , Jörg Simon , Tom Clarke , David J. Richardson , Julea N. Butt Journal Of The American Chemical Society 137 (8), 3059 - 3068 DOI: 10.1021/ja512941j PMID: 25658043 Diamond Proposal Number(s): [7641] Open Access Show Abstract ▼ Abstract: Cytochrome c nitrite reductases perform a key step in the biogeochemical N-cycle by catalyzing the six-electron reduction of nitrite to ammonium. These multiheme cytochromes contain a number of His/His ligated c-hemes for electron transfer and a structurally differentiated heme that provides the catalytic center. The catalytic heme has proximal ligation from lysine, or histidine, and an exchangeable distal ligand bound within a pocket that includes a conserved histidine. Here we describe properties of a penta-heme cytochrome c nitrite reductase in which the distal His has been substituted by Asn. The variant is unable to catalyze nitrite reduction despite retaining the ability to reduce a proposed intermediate in that process, namely, hydroxylamine. A combination of electrochemical, structural and spectroscopic studies reveals that the variant enzyme simultaneously binds nitrite and electrons at the catalytic heme. As a consequence the distal His is proposed to play a key role in orienting the nitrite for NO bond cleavage. The electrochemical experiments also reveal that the distal His facilitates rapid nitrite binding to the catalytic heme of the native enzyme. Finally it is noted that the thermodynamic descriptions of nitrite- and electron-binding to the active site of the variant enzyme are modulated by the prevailing oxidation states of the His/His ligated hemes. This behavior is likely to be displayed by other multicentered redox enzymes such that there are wide implications for considering the determinants of catalytic activity in this important and varied group of oxidoreductases.	Mar 2015
I02-Macromolecular Crystallography	Characterization of the active site and calcium binding in cytochrome c nitrite reductases Andrew m. Hemmings , Colin Lockwood , Thomas Clarke , Julea N. Butt , David Richardson Biochemical Society Transactions 39 (6), 1871-1875 DOI: 10.1042/BST20110731 PMID: 22103542 Diamond Proposal Number(s): [303] Show Abstract ▼ Abstract: The decahaem homodimeric cytochrome c nitrite reductase (NrfA) is expressed within the periplasm of a wide range of Gamma-, Delta- and Epsilon-proteobacteria and is responsible for the six-electron reduction of nitrite to ammonia. This allows nitrite to be used as a terminal electron acceptor, facilitating anaerobic respiration while allowing nitrogen to remain in a biologically available form. NrfA has also been reported to reduce nitric oxide (a reaction intermediate) and sulfite to ammonia and sulfide respectively, suggesting a potential secondary role as a detoxification enzyme. The protein sequences and crystal structures of NrfA from different bacteria and the closely related octahaem nitrite reductase from Thioalkalivibrio nitratireducens (TvNir) reveal that these enzymes are homologous. The NrfA proteins contain five covalently attached haem groups, four of which are bis-histidine-co-ordinated, with the proximal histidine being provided by the highly conserved CXXCH motif. These haems are responsible for intraprotein electron transfer. The remaining haem is the site for nitrite reduction, which is ligated by a novel lysine residue provided by a CXXCK haem-binding motif. The TvNir nitrite reductase has five haems that are structurally similar to those of NrfA and three extra bis-histidine-coordinated haems that precede the NrfA conserved region. The present review compares the protein sequences and structures of NrfA and TvNir and discusses the subtle differences related to active-site architecture and Ca2+ binding that may have an impact on substrate reduction.	Dec 2011
I02-Macromolecular Crystallography	Kinetic and thermodynamic resolution of the interactions between sulfite and the pentahaem cytochrome NrfA from Escherichia coli Andrew m. Hemmings , Gemma Kemp , Thomas A. Clarke , Sophie J. Marritt , Colin Lockwood , Susannah R. Poock , David J. Richardson , Miles R. Cheeseman , Julea N. Butt Biochemical Journal 431 (1), 73-80 DOI: 10.1042/BJ20100866 PMID: 20629638 Diamond Proposal Number(s): [303] Show Abstract ▼ Abstract: NrfA is a pentahaem cytochrome present in a wide-range of ?-, ?- and ?-proteobacteria. Its nitrite and nitric oxide reductase activities have been studied extensively and contribute to respiratory nitrite ammonification and nitric oxide detoxification respectively. Sulfite is a third substrate for NrfA that may be encountered in the micro-oxic environments where nrfA is expressed. Consequently, we have performed quantitative kinetic and thermodynamic studies of the interactions between sulfite and Escherichia coli NrfA to provide a biochemical framework from which to consider their possible cellular consequences. A combination of voltammetric, spectroscopic and crystallographic analyses define dissociation constants for sulfite binding to NrfA in oxidized (~54 ?M), semi-reduced (~145 ?M) and reduced (~180 ?M) states that are comparable with each other, and the Km (~70 ?M) for sulfite reduction at pH 7. Under comparable conditions Km values of ~22 and ~300 ?M describe nitrite and nitric oxide reduction respectively, whereas the affinities of nitrate and thiocyanate for NrfA fall more than 50-fold on enzyme reduction. These results are discussed in terms of the nature of sulfite co-ordination within the active site of NrfA and their implications for the cellular activity of NrfA.	Oct 2010

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