Kinetic and thermodynamic resolution of the interactions between sulfite and the pentahaem cytochrome NrfA from Escherichia coli.
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Kemp GL, Clarke TA, Marritt SJ, Lockwood C, Poock SR, Hemmings AM, Richardson DJ, Cheesman MR, Butt JN
Kinetic and thermodynamic resolution of the interactions between sulfite and the pentahaem cytochrome NrfA from Escherichia coli.
Biochem J. 2010 Oct 1;431(1):73-80. doi: 10.1042/BJ20100866.
- PubMed ID
- 20629638 [ View in PubMed]
- Abstract
NrfA is a pentahaem cytochrome present in a wide-range of gamma-, delta- and epsilon-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 muM), semi-reduced (~145 muM) and reduced (~180 muM) states that are comparable with each other, and the Km (~70 muM) for sulfite reduction at pH 7. Under comparable conditions Km values of ~22 and ~300 muM 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.