vitamin-k-semiquinone-radical and duroquinol

We have compared the steady-state kinetics of wild-type nitrate reductase A and two mutant forms with altered beta subunits. To mimic conditions in vivo as closely as possible, we used analogues of the physiological quinols as electron donors and membranes with overexpressed nitrate reductase A in preference to a purified alpha beta gamma complex. With the wild-type enzyme both menadiol and duroquinol supply their electrons for the reduction of nitrate at rates that depend on the square of the quinol concentration, menadiol having the higher catalytic constant. The results as a whole are consistent with a substituted-enzyme mechanism for the reduction of nitrate by the quinols. Kinetic experiments suggest that duroquinol and menadiol deliver their electrons at different sites on nitrate reductase, with cross-inhibition. Menadiol inhibits the duroquinol reaction strongly, suggesting that menaquinol may be the preferred substrate in vivo. To examine whether electron transfer from menadiol and duroquinol for nitrate reduction requires the presence of all of the Fe-S centres, we have studied the steady-state kinetics of mutants with beta subunits that lack an Fe-S centre. The loss of the highest-potential Fe-S centre results in an enzyme without menadiol activity, but retaining duroquinol activity; the kinetic parameters are within a factor of two of those of the wild-type enzyme, indicating that this centre is not required for the duroquinol activity. The loss of a low-potential Fe-S centre affects the activity with both quinols: the enzyme is still active but the catalytic constants for both quinols are decreased by about 75%, indicating that this centre is important but not essential for the activity. The existence of a specific site of reaction on nitrate reductase for each quinol, together with the differences in the effects on the two quinols produced by the loss of the Fe-S centre of +80 mV, suggests that the pathways for transfer of electrons from duroquinol and menadiol are not identical.

Topics: Escherichia coli; Hydroquinones; Kinetics; Mutation; Nitrate Reductase; Nitrate Reductases; Vitamin K

The quinol oxidase appears to be mainly responsible for the oxidation of the bacterial MKH2 in Bacillus subtilis W23 growing with either glucose or succinate. The activity of the enzyme was maximum with dimethylnaphthoquinol, a water-soluble analogue of the bacterial menaquinol. Menadiol or duroquinol were less actively respired, and naphthoquinol was not oxidized at all. After fourtyfold purification the isolated enzyme contained 5.3 mumol cytochrome aa3 per gram of protein and negligible amounts of cytochrome b and c. The turnover number based on cytochrome aa3 was about 10(3) electrons.s-1 at pH 7 and 37 degrees C. The preparation consisted mainly of a M(r) 57,000 and a M(r) 36,000 polypeptide. The N-terminal amino acid sequence of the latter polypeptide differed from that predicted by the qoxA gene of B. subtilis strain 168 (Santana et al. 1992), in that asp-14 predicted by qoxA was missing in the M(r) 36,000 polypeptide.

Topics: Amino Acid Sequence; Bacillus subtilis; Chromatography, Ion Exchange; Electron Transport Complex IV; Genes, Bacterial; Hydroquinones; Molecular Sequence Data; Naphthoquinones; Oxidation-Reduction; Vitamin K