Aldehyde oxidoreductase activity in Desulfovibrio alaskensis NCIMB 13491

Abstract

A novel molybdenum iron-sulfur-containing aldehyde oxidoreductase (AOR) belonging to the xanthine oxidase family was isolated and characterized from the sulfate-reducing bacterium Desulfovibrio alaskensis NCIMB 13491, a strain 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. . Three different EPR signals were recorded upon long reduction of the protein with excess dithionite: an almost axial signal split by hyperfine interaction with one proton associated with Mo(V) species and two rhombic signals with EPR parameters and relaxation behavior typical of [2Fe±2S] clusters termed Fe/S I and Fe/S II, respectively. EPR results reveal the existence of magnetic interactions between Mo(V) and one of the Fe/S clusters, as well as between the two Fe/S clusters. Redox titration monitored by EPR yielded midpoint redox potentials of 2275 and 2325 mV for the Fe/S I and Fe/S II, 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 centers, 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.

Correlating EPR and X-ray structural analysis of arsenite-inhibited forms of aldehyde oxidoreductase

“…Both EPR signals A and B show temperature-dependent splittings produced by magnetic coupling with FeS I, as observed in dithionitereduced samples of the enzymes of the XO family [25]. Magnetic interactions depending on temperature occur when one of the species (FeS I) of the interacting pair has a relaxation rate (T 1 ) faster than the other one [Mo(V)], which produces an enhancement of the relaxation rate of the slowly relaxing paramagnetic center and, consequently, a temperature dependence of the splitting of the resonance lines [25,26,43,44]. The parameters and the temperature dependence of the EPR signals associated with the FeS centers in samples with signal B are similar to those obtained in reduced samples of as-purified DgAOR, indicating no structural Air exposure of the dithionite-reduced samples of both samples (signals A and B) oxidizes completely the FeS clusters to a diamagnetic state but leaves the Mo(V) species reduced.…”

“…FeS I, the proximal center, is closer to the Mo site and is buried inside the protein, whereas FeS II, the distal center, is situated near the surface of the protein. Both iron-sulfur centers and a fraction of the molybdenum ions are paramagnetic in the reduced state of the protein and, in addition, show intercenter magnetic couplings [25][26][27]. The currently accepted mechanism implies substrate interaction with the Mo center, which is then reduced from Mo(VI) to Mo(IV), followed by a two electron transfer to an external electron acceptor, in a process mediated by the two iron-sulfur centers.…”

Mo and W bis-MGD enzymes: nitrate reductases and formate dehydrogenases

“…In the same line, D. alaskensis Fdh can incorporate both metal ions in a medium with traces of both metals. In the same medium where D. alaskensis Fdh was obtained, an aldehyde oxidoreductase (AOR), almost identical to the Mo-AORs from SRB, contains only Mo [53]. In contrast, D. gigas Fdh [3] and Fdh enzymes from Syntrophobacter fumaroxidans incorporate W [5], despite their growth with enough Mo in the medium.…”

Kinetic, Structural, and EPR Studies Reveal That Aldehyde Oxidoreductase from Desulfovibrio gigas Does Not Need a Sulfido Ligand for Catalysis and Give Evidence for a Direct Mo−C Interaction in a Biological System

“…20 In addition, the Mo(V) EPR signals associated with desulfo forms of XO, such as the "slow"-type signal and the signal obtained upon ethylene glycol (EDO) addition, were also observed. [20][21][22][23] On this basis, it was assumed that AORs from SRB and XO family enzymes have similar active sites and that both would experience the same changes upon reaction with substrates, inhibitors, and reducing agents. Because the structural data of DgAOR did not show the sulfido ligand coordinated to Mo, it was assumed that the enzyme crystallizes in the desulfo inactive form (Figure 1b).…”