Studies on the Redox Centers of the Terminal Oxidase fromDesulfovibrio gigas and Evidence for Its Interaction with Rubredoxin

Abstract

Rubredoxin-oxygen oxidoreductase (ROO) is the final component of a soluble electron transfer chain that couples NADH oxidation to oxygen consumption in the anaerobic sulfate reducer Desulfovibrio gigas. It is an 86-kDa homodimeric flavohemeprotein containing two FAD molecules, one mesoheme IX, and one Fe-uroporphyrin I per monomer, capable of fully reducing oxygen to water. EPR studies on the native enzyme reveal two components with g values at ϳ2.46, 2.29, and 1.89, which are assigned to low spin hemes and are similar to the EPR features of P-450 hemes, suggesting that ROO hemes have a cysteinyl axial ligation. At pH 7.6, the flavin redox transitions occur at 0 ؎ 15 mV for the quinone/semiquinone couple and at ؊130 ؎ 15 mV for the semiquinone/hydroquinone couple; the hemes reduction potential is ؊350 ؎ 15 mV. Spectroscopic studies provided unequivocal evidence that the flavins are the electron acceptor centers from rubredoxin, and that their reduction proceed through an anionic semiquinone radical. The reaction with oxygen occurs in the flavin moiety. These data are strongly corroborated by the finding that rubredoxin and ROO are located in the same polycistronic unit of D. gigas genome. For the first time, a clear role for a rubredoxin in a sulfate-reducing bacterium is presented.Despite the fact that they are still being considered as strict anaerobes, sulfate reducing bacteria when exposed to oxygen are capable of surviving (1, 2) as well as of taking advantage of its presence in terms of energy conservation (3, 4). In the presence of oxygen, the sulfate reducer Desulfovibrio gigas, uses internal reserves of polyglucose, which is metabolized by the Embden-Meyerhof-Parnas pathway thus generating NADH and ATP (3). In this bacterium a three-component soluble electron transfer chain couples NADH oxidation with oxygen reduction to water, allowing simultaneously for NAD ϩ regeneration and oxygen utilization. The proteins involved are NADH:rubredoxin oxidoreductase (NRO) 1 (5), rubredoxin (Rd) (6), and rubredoxin:oxygen oxidoreductase (ROO) (7), as shown in Scheme I.NRO, the first component of this pathway, contains two subunits of 27 and 32 kDa and is a flavoprotein containing four flavin groups (FMN and FAD) per molecule with reduction potentials (Fl ox /Fl red ) of Ϫ295 and Ϫ325 mV, at pH 7.6. This NADH oxidase reduces D. gigas Rd, a 6-kDa iron protein, with a reduction potential of 0 Ϯ 5 mV. NRO is highly specific toward this redox protein (5). D. gigas desulforedoxin (8), Desulfovibrio desulfuricans Rd (9), or even Desulfovibrio vulgaris Rd (10) are hardly reduced by NRO (5), despite the similarity between the reduction potentials of these proteins. D. gigas Rd was also found to be necessary for the coupling of NRO to the final component of the chain, ROO (7). This enzyme can be seen as a true terminal oxidase since it is capable of catalyzing the four-electron reduction of oxygen to water without the formation of partially reduced oxygen species (7). It is an 86-kDa homodimeric flavohemeprotein, containing tw...

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“…Likewise, rub2 forms an operon with the CPE0781 gene, encoding a hypothetical flavoprotein. The product shares 29 % identity and 47 % similarity with the rubredoxin-oxygen-oxidoreductase (Roo) from Desulfovibrio gigas, which is believed to catalyse the reduction of dioxygen to water (Gomes et al, 1997;. Two putative regulatory genes (CPE0776 and CPE0779) are located upstream of two rubredoxinencoding genes.…”

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