The hemes of Escherichia coli nitrate reductase A (NarGHI): potentiometric effects of inhibitor binding to narI.
We have potentiometrically characterized the two hemes of Escherichia coli nitrate reductase A (NarGHI) using EPR and optical spectroscopy. NarGHI contains two hemes, a low-potential heme b(L) (E(m,7) = 20 mV; g(z)() = 3.36) and a high-potential heme b(H) (E(m, 7) = 120 mV; g(z)() = 3.76). Potentiometric analyses of the g(z)() features of the heme EPR spectra indicate that the E(m,7) values of both hemes are sensitive to the menaquinol analogue 2-n-heptyl-4-hydroxyquinoline N-oxide (HOQNO). This inhibitor causes a potential-inversion of the two hemes (for heme b(L), E(m,7) = 120 mV; for heme b(H), E(m,7) = 60 mV). This effect is corroborated by optical spectroscopy of a heme b(H)-deficient mutant (NarGHI(H56R)) in which the heme b(L) undergoes a DeltaE(m,7) of 70 mV in the presence of HOQNO. Another potent inhibitor of NarGHI, stigmatellin, elicits a moderate heme b(L) DeltaE(m,7) of 30 mV, but has no detectable effect on heme b(H). No effect is elicited by either inhibitor on the line shape or the E(m,7) values of the [3Fe-4S] cluster coordinated by NarH. When NarI is expressed in the absence of NarGH [NarI(DeltaGH)], two hemes are detected in potentiometric titrations with E(m,7) values of 37 mV (heme b(L); g(z)() = 3.15) and -178 mV (heme b(H); g(z)() = 2.92), suggesting that heme b(H) may be exposed to the aqueous milieu in the absence of NarGH. The identity of these hemes was confirmed by recording EPR spectra of NarI(DeltaGH)(H56R). HOQNO binding titrations followed by fluorescence spectroscopy suggest that in both NarGHI and NarI(DeltaGH), this inhibitor binds to a single high-affinity site with a K(d) of approximately 0.2 microM. These data support a functional model for NarGHI in which a single dissociable quinol binding site is associated with heme b(L) and is located toward the periplasmic side of NarI.
Rothery
R A
RA
Medical Research Council of Canada Group in the Molecular Biology of Membrane Proteins, Department of Biochemistry, 474 Medical Sciences Building, University of Alberta, Edmonton, Alberta T6G 2H7, Canada,
Blasco
F
F
Magalon
A
A
Asso
M
M
Weiner
J H
JH
D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D004791:Enzyme Inhibitors / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D005453:Fluorescence; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D011090:Polyenes / Q000378:metabolism; D011199:Potentiometry
D004791:Enzyme Inhibitors; D011090:Polyenes; C041573:stigmatellin; D009565:Nitrate Reductases; D050901:Nitrate Reductase