Oxidation-reduction potentials of flavin and Mo-pterin centers in assimilatory nitrate reductase: variation with pH.
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.(ABSTRACT TRUNCATED AT 250 WORDS)
Kay
C J
CJ
Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, Tampa 33612.
Solomonson
L P
LP
Barber
M J
MJ
D002708:Chlorella / Q000201:enzymology*; D002942:Circular Dichroism; D003067:Coenzymes; D004578:Electron Spin Resonance Spectroscopy; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008667:Metalloproteins / Q000378:metabolism*; D008982:Molybdenum / Q000378:metabolism; D000090006:Molybdenum Cofactors; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000378:metabolism*; D010084:Oxidation-Reduction; D011199:Potentiometry; D011621:Pteridines / Q000378:metabolism*
D003067:Coenzymes; D005182:Flavin-Adenine Dinucleotide; D008667:Metalloproteins; D008982:Molybdenum; D000090006:Molybdenum Cofactors; D050901:Nitrate Reductase; D009565:Nitrate Reductases; D011621:Pteridines; C022261:molybdopterin