Oxidation-reduction potentials of flavin and Mo-pterin centers in assimilatory nitrate reductase: variation with pH

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Abstract
Potentiometric titrations of assimilatory nitrate reductase from Chlorella vulgaris were performed within the pH range 6.0-9.0. Mo(V) was measured by room temperature EPR spectroscopy while the reduction state of FAD was monitored by CD spectroscopy. Between pH 6 and 8.5, the line shape of the Mo(V) EPR signal was constant, exhibiting superhyperfine coupling to a single, exchangeable proton. Potentiometric titrations indicated the Em values for the Mo(VI)/Mo(V) (+61 mV, pH 6) and Mo(V)/Mo(IV) (+35 mV, pH 6) couples decreased with increasing pH by approximately -59 mV/pH unit, consistent with the uptake of a single proton upon reduction of Mo(VI) to Mo(V) and Mo(V) to Mo(IV). The pKa values for the dissociation of these redox-coupled protons appeared to lie outside the pH range studied: pKO(MoVI), pKO(MoV) 9. The Em (n = 2) for FAD (-250 mV, pH 7) varied by approximately -30 mV/pH unit within the pH range 6.0-9.0. Low-temperature EPR potentiometry at the extreme pH values indicated less than 0.5% conversion of FAD to the semiquinone form at the midpoint of the titrations. In contrast, NADH-reduced enzyme exhibited approximately 3-5% of the FAD in the semiquinone form, present as the anionic (FAD.-) species, the spectrum characterized by a line width of 1.3 mT at both pH 6.0 and 9.0. These results were consistent with the following reduction scheme: FAD + e- .fwdarw. FAD.- + e- + H+ .fwdarw. FADH-, with pKr(FADH-) of >9.5, a pH-independent value for Em (FAD/FAD.-) of <-458 mV, and a pH dependence of the Em for the FAD.-/FADH- redox couple of -59 mV/pH unit. Combining these results with data previously published for the heme center [Kay, C.J., Solomonson, L. P., and Barber, M.J. (1986) J. Biol. Chem. 261, 5799-5802], the uptake of four protons appears to be coupled to the oxidation-reduction reactions of Chlorella nitrate reductase at room temperature: one stoichiometrically coupled to FAD.- reduction, one weakly coupled to heme reduction, and two independent coupled to reduction of Mo(VI) and Mo(V). At all pH''s examined, nitrate reductase activity remained thermodynamically favorable.