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

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 → Gln variant enzyme at 3.1-Å resolution rules out any major structural changes compared with the wild-type enzyme. The oxidation-reduction 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.

Nonphotochemical Reduction of the Plastoquinone Pool in Sunflower Leaves Originates from Chlororespiration1

We investigated the relationship between nonphotochemical plastoquinone reduction and chlororespiration in leaves of growth-chamber-grown sunflower (Helianthus annuus L.). Following a short induction period, leaves of previously illuminated sunflower showed a substantially increased level of minimal fluorescence following a light-to-dark transition. This increase in minimal fluorescence was reversed by far-red illumination, inhibited by rotenone or photooxidative methyl viologen treatment, and stimulated by fumigation with CO. Using flash-induced electrochromic absorption-change measurements, we observed that the capacity of sunflower to reduce plastoquinone in the dark influenced the activation state of the chloroplast ATP synthase, although chlororespiratory transmembrane electrochemical potential formation alone does not fully explain our observations. We have added several important new observations to the work of others, forming, to our knowledge, the first strong experimental evidence that chlororespiratory, nonphotochemical plastoquinone reduction and plastoquinol oxidation occur in the chloroplasts of higher plants. We have introduced procedures for monitoring and manipulating chlorores-piratory activity in leaves that will be important in subsequent work aimed at defining the pathway and function of this dark electron flux in higher plant chloroplasts.