Kinetic and thermodynamic resolution of the interactions between sulfite and the pentahaem cytochrome NrfA from Escherichia coli

Kemp, Gemma L., Clarke, Thomas A., Marritt, Sophie J., Lockwood, Colin, Poock, Susannah R., Hemmings, Andrew M., Richardson, David J., Cheesman, Myles R. and Butt, Julea N. (2010) Kinetic and thermodynamic resolution of the interactions between sulfite and the pentahaem cytochrome NrfA from Escherichia coli. Biochemical Journal, 431 (1). pp. 73-80. ISSN 0264-6021

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Abstract

NrfA is a pentahaem cytochrome present in a wide-range of ?-, d- and e-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.

Item Type: Article
Faculty \ School: Faculty of Science > School of Biological Sciences
Faculty of Science > School of Chemistry
Depositing User: Vishal Gautam
Date Deposited: 12 Sep 2011 10:52
Last Modified: 21 Apr 2020 17:00
URI: https://ueaeprints.uea.ac.uk/id/eprint/20547
DOI: 10.1042/BJ20100866

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