The hemes of Escherichia coli nitrate reductase A (NarGHI): Potentiometriceffects 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 con tains two hemes, a low potential heme b(L) (E-m,E-7 = 20 mV; g(z) = 3.36) a nd a high-potential heme b(H) (E-m,E-7 = 120 mV; g(z) = 3.76). Potentiometr ic analyses of the g(z) features of the heme EPR spectra indicate that the E-m,E-7 values of both hemes are sensitive to the menaquinol analogue 2-n-h eptyl-4-hydroxyquinoline N-oxide (HOQNO). This inhibitor causes a potential -inversion of the two hemes (for heme b(L), E-m,E-7 = 120 mV; for heme b(H) , E-m,E-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) undergoe s a Delta E-m,E-7 Of 70 mV in the presence of HOQNO. Another potent inhibit or of NarGHI, stigmatellin, elicits a moderate heme b(L) Delta E-m,E-7 Of 3 0 mV, but has no detectable effect on heme b(H). No effect is elicited by e ither inhibitor on the line shape or the E-m,E-7 values of the [3Fe-4S] clu ster coordinated by NarH, When NarI is expressed in the absence of NarGH [N arI(Delta GH)], two hemes are detected in potentiometric titrations with E- m,E-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 recordi ng EPR spectra of NarI(Delta GH)(H56R). HOQNO binding titrations followed b y fluorescence spectroscopy suggest that in both NarGHI and NarI(Delta GH), this inhibitor binds to a single high-affinity site with a K-d Of approxim ately 0.2 mu M 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.