Kinetic and thermodynamic resolution of the interactions between sulphite and the penta-heme cytochrome, NrfA, from Escherichia coli.

Gemma L Kemp , Thomas A Clarke , Sophie J Marritt , Colin Lockwood , Susannah R Poock , Andrew M Hemmings , David J Richardson , Myles Cheesman , Julea N Butt (1)

1 School of Chemistry

NrfA is a pentaheme 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. Sulphite is a third substrate for NrfA that may be encountered in the microoxic environments where nrfA is expressed. Consequently, we have performed quantitative kinetic and thermodynamic studies of the interactions between sulphite and E. 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 sulphite binding to NrfA in oxidised (ca. 54 µM), semi-reduced (ca. 145 µM) and reduced (ca. 180 µM) states that are comparable to each other and to the KM (ca. 70 µM) for sulphite reduction at pH 7. Under comparable conditions KM values of ca. 22 and 300 µM describe nitrite and nitric oxide reduction respectively, while the affinities of nitrate and thiocyanate for NrfA drop more than 50-fold on enzyme reduction. These results are discussed in terms of the nature of sulphite coordination within the active site of NrfA and their implications for the cellular activity of NrfA.

Mots clés: Life Sciences

Gemma L Kemp, Thomas A Clarke, Sophie J Marritt, Colin Lockwood, Susannah R Poock, et al.. Kinetic and thermodynamic resolution of the interactions between sulphite and the penta-heme cytochrome, NrfA, from Escherichia coli.. Biochemical Journal, 2010, 431 (1), pp.73-80. ⟨10.1042/BJ20100866⟩. ⟨hal-00517256⟩