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

DOI: 10.1042/BJ20100866 DOI Help
PMID: 20629638 PMID Help

Authors: Andrew m. Hemmings (Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, U.K.) , Gemma Kemp (Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, U.K.) , Thomas A. Clarke (School of Biological Sciences, University of East Anglia) , Sophie J. Marritt (Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, U.K.) , Colin Lockwood (John Innes Centre, U.K.) , Susannah R. Poock (School of Biological Sciences, University of East Anglia) , David J. Richardson (School of Biological Sciences, University of East Anglia, U.K.) , Miles R. Cheeseman (Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia) , Julea N. Butt (Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Biochemical Journal , VOL 431 (1) , PAGES 73-80

State: Published (Approved)
Published: October 2010
Diamond Proposal Number(s): 303

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.

Journal Keywords: Enzyme; Structure; Mechanism

Subject Areas: Biology and Bio-materials


Instruments: I02-Macromolecular Crystallography

Other Facilities: ESRF

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