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

• Published in: Biochemistry (Easton) 1990-12, Vol.29 (48), p.10823-10828
• Main Authors: Kay, Christopher J, Solomonson, Larry P, Barber, Michael J
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Chlorella - enzymology; Circular Dichroism; Coenzymes; Electron Spin Resonance Spectroscopy; Enzymes and enzyme inhibitors; Flavin-Adenine Dinucleotide - metabolism; Fundamental and applied biological sciences. Psychology; Hydrogen-Ion Concentration; Metalloproteins - metabolism; Molybdenum - metabolism; Nitrate Reductase; Nitrate Reductases - metabolism; Oxidation-Reduction; Oxidoreductases; Potentiometry; Pteridines - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00500a015

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) less than 5.5; pKr(MoV), pKr(MoIV) greater than 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.