title: Kinetic and thermodynamic resolution of the interactions between sulfite and the pentahaem cytochrome NrfA from Escherichia coli.
abstract: NrfA is a pentahaem cytochrome present in a wide-range of γ-, δ- and ε-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 μM), semi-reduced (~145 μM) and reduced (~180 μM) states that are comparable with each other, and the Km (~70 μM) for sulfite reduction at pH 7. Under comparable conditions Km values of ~22 and ~300 μM 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.
authors.affiliation: Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, UK.
authors.forename: Gemma L
authors.initials: GL
authors.lastname: Kemp
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Clarke
authors.forename: Sophie J
authors.initials: SJ
authors.lastname: Marritt
authors.forename: Colin
authors.initials: C
authors.lastname: Lockwood
authors.forename: Susannah R
authors.initials: SR
authors.lastname: Poock
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Hemmings
authors.forename: David J
authors.initials: DJ
authors.lastname: Richardson
authors.forename: Myles R
authors.initials: MR
authors.lastname: Cheesman
authors.forename: Julea N
authors.initials: JN
authors.lastname: Butt
mesh_terms: D020134:Catalytic Domain; D003574:Cytochrome c Group / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011487:Protein Conformation; D013816:Thermodynamics
chemical_list: D003574:Cytochrome c Group; D006418:Heme; C022302:cytochrome C-552