Circular dichroism and potentiometry of FAD, heme and Mo-pterin prosthetic groups of assimilatory nitrate reductase.
Oxidation-reduction midpoint potentials for flavin, heme, and molybdenum-pterin prosthetic groups of assimilatory nitrate reductase (NR) from Chlorella vulgaris were measured at room temperature by using CD and EPR potentiometry. The CD changes accompanying reduction of each prosthetic group were determined by using enzyme fragments containing either FAD or heme and molybdenum prosthetic groups, obtained by limited proteolysis, and by poising the enzyme at various redox potentials in the presence of dye mediators. Limited proteolysis did not appear to alter the environment of the prosthetic groups, as judged by their CD spectra. Also, CD potentiometric titration of FAD in intact NR (Em' = -272 mV, n = 2) gave a similar value (Em' = -286 mV) to the FAD of the flavin-containing proteolytic domain, determined by visible spectroscopy. Less than 1% of the flavin semiquinone was detected by EPR spectroscopy, indicating that Em' (FAD/FAD.-) may be more than 200 mV lower than Em' (FAD.-/FADH-). Reduction of heme resulted in splitting of both Soret and alpha CD bands into couplets. The heme Em' was -162 mV (n = 1) determined by both CD and visible spectroscopy. Reduction of Mo-pterin was followed by CD at 333 nm, and Mo(V) was monitored by room temperature EPR spectroscopy. Most of the change in the Mo-pterin CD spectrum was due to the Mo(VI)/Mo(V) transition. The Em' values determined for Mo(VI)/Mo(V) were +26 mV by CD and +16 mV by EPR, whereas Mo(V)/Mo(IV) values were -40 mV by CD and -26 mV by EPR.(ABSTRACT TRUNCATED AT 250 WORDS)
Kay
C J
CJ
Department of Biochemistry, College of Medicine, University of South Florida, Tampa 33612.
Barber
M J
MJ
Solomonson
L P
LP
D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D005182:Flavin-Adenine Dinucleotide; D006418:Heme; D008982:Molybdenum; D050901:Nitrate Reductase; D009565:Nitrate Reductases; D010084:Oxidation-Reduction; D011199:Potentiometry; D011622:Pterins; D013057:Spectrum Analysis
D011622:Pterins; D005182:Flavin-Adenine Dinucleotide; D006418:Heme; D008982:Molybdenum; D009565:Nitrate Reductases; D050901:Nitrate Reductase