Aldehyde oxidoreductase activity in Desulfovibrio alaskensis NCIMB 13491 -EPR assignment of the proximal [2Fe-2S] cluster to the Mo site

A novel molybdenum iron-sulfur-containing aldehyde oxidoreductase (AOR) bel onging to the xanthine oxidase family was isolated and characterized from t he sulfate-reducing bacterium Desulfovibrio alaskensis NCIMB 13491, a strai n isolated from a soured oil reservoir in Purdu Bay, Alaska. D. alaskensis AOR is closely related to other AORs isolated from the Desulfovibrio genus. The protein is a 97-kDa homodimer, with 0.6 +/- 0.1 Mo, 3.6 +/- 0.1 Fe and 0.9 +/- 0.1 pterin cytosine dinucleotides per monomer. The enzyme catalyse s the oxidation of aldehydes to their carboxylic acid form, following simpl e Michaelis-Menten kinetics, with the following parameters (for benzaldehyd e): K-m(app) = 6.65 mu m; V-app = 13.12 mu m.min(-1); k(cat)(app) = 0.96 s( -1). Three different EPR signals were recorded upon long reduction of the p rotein with excess dithionite: an almost axial signal split by hyperfine in teraction with one proton associated with Mo(V) species and two rhombic sig nals with EPR parameters and relaxation behavior typical of [2Fe-2S] cluste rs termed Fe/S I and Fe/S II, respectively. EPR results reveal the existenc e of magnetic interactions between Mo(V) and one of the Fe/S clusters, as w ell as between the two Fe/S clusters. Redox titration monitored by EPR yiel ded midpoint redox potentials of -275 and -325 mV for the Fe/S I and Fe/S I I, respectively. The redox potential gap between the two clusters is large enough to obtain differentiated populations of these paramagnetic centers. This fact, together with the observed interactions among paramagnetic cente rs, was used to assign the EPR-distinguishable Fe/S I and Fe/S II to those seen in the reported crystal structures of homologous enzymes.