Essential role of Glu-C66 for menaquinol oxidation indicates transmembrane electrochemical potential generation by Wolinella succinogenes fumarate reductase

Abstract

Quinol:fumarate reductase (QFR) is a membrane protein complex that couples the reduction of fumarate to succinate to the oxidation of quinol to quinone, in a reaction opposite to that catalyzed by the related enzyme succinate:quinone reductase (succinate dehydrogenase). In the previously determined structure of QFR from Wolinella succinogenes, the site of fumarate reduction in the flavoprotein subunit A of the enzyme was identified, but the site of menaquinol oxidation was not. In the crystal structure, the acidic residue Glu-66 of the membrane spanning, diheme-containing subunit C lines a cavity that could be occupied by the substrate menaquinol. Here we describe that, after replacement of Glu-C66 with Gln by site-directed mutagenesis, the resulting mutant is unable to grow on fumarate and the purified enzyme lacks quinol oxidation activity. X-ray crystal structure analysis of the Glu-C66 3 Gln variant enzyme at 3.1-Å resolution rules out any major structural changes compared with the wild-type enzyme. The oxidationreduction potentials of the heme groups are not significantly affected. We conclude that Glu-C66 is an essential constituent of the menaquinol oxidation site. Because Glu-C66 is oriented toward a cavity leading to the periplasm, the release of two protons on menaquinol oxidation is expected to occur to the periplasm, whereas the uptake of two protons on fumarate reduction occurs from the cytoplasm. Thus our results indicate that the reaction catalyzed by W. succinogenes QFR generates a transmembrane electrochemical potential.

Experimental support for the “E pathway hypothesis” of coupled transmembrane e – and H + transfer in dihemic quinol:fumarate reductase

“…Upon solubilization of the isolated membranes with Triton X-100 (14,22,23), however, the activities of fumarate reduction by DMNH 2 for the Glu-C180 variants were detectable and corresponded to approximately 1͞10 of that of the wild-type enzyme. In this respect, the Glu-C180 QFR variants differ significantly from the Glu-C66 3 Gln variant (16). The detergentsolubilized Glu-C180 variants are similar to the wild-type enzyme considering the fraction (1͞2) of the dithionite-reducible heme b that could be reduced by DMNH 2 ( Table 1).…”

“…The mutant strains FrdC-E180Q and FrdC-E180I are compared in Table 1 to the wild type and the previously characterized strain FrdC-E66Q, where the essential residue Glu-C66 (Fig. 1b) at the menaquinol oxidation site was replaced with a Gln (16). The mutants did not grow with fumarate as the terminal electron acceptor; however, they did grow when nitrate (and brain-heart infusion broth) replaced fumarate.…”

“…Although electrophysiological experiments performed with inverted vesicles and proteoliposomes containing QFR demonstrated that the reaction catalyzed by the diheme-containing QFR from W. succinogenes is not directly associated with the generation of a transmembrane electrochemical proton potential (11)(12)(13), the three-dimensional structure of this membrane protein complex, initially solved at 2.2-Å resolution (14), revealed locations of the active sites of fumarate reduction (15) and of menaquinol oxidation (16) that are oriented toward opposite sides of the membrane (Fig. 1b).…”

Structural and Biochemical Characterization of a Quinol Binding Site of Escherichia coli Nitrate Reductase A

“…5b) where both hemes are exposed is a good candidate for a secondary quinone reactive site. On the other hand, the apolar pocket A does not contain any ionizable group as found in a number of Q-sites, including Q P in E. coli fumarate reductase (35), Q D in Wolinella succinogenes fumarate reductase (36), and Q D in E. coli FdnGHI (4). This observation suggests that the hypothetical secondary Q-site may be important for structural integrity of NarI with possible electron transfer but not proton translocation activities.…”

Characterization of the Menaquinone Reduction Site in the Diheme Cytochrome b Membrane Anchor of Wolinella succinogenes NiFe-hydrogenase

“…In addition to the cytochrome b, both enzymes contain two further hydrophilic subunits that are located at the inner surface of the membrane where the interconversion of fumarate and succinate is coupled to proton uptake or release. It was proposed that a menaquinone reduction site is located near the outer surface of the cytochrome b subunit in both enzymes implying that both heme b groups are essential for transmembrane electron transfer (41,42). Assuming that protons involved in the redox reactions of menaquinone are exchanged at the outer side of the membrane, succinate oxidation by menaquinone as well as fumarate reduction by menaquinol would be electrogenic processes.…”