Substrate control of internal electron transfer in bacterial nitric-oxide reductase

Abstract

Nitric -oxide reductase (NOR) from Paracoccus denitrificans catalyzes the reduction of nitric oxide (NO) to nitrous oxide (N(2)O) (2NO + 2H(+) + 2e(-) -->N(2)O + H(2)O) by a poorly understood mechanism. NOR contains two low spin hemes c and b, one high spin heme b(3), and a non-heme iron Fe(B). Here, we have studied the reaction between fully reduced NOR and NO using the "flow-flash" technique. Fully (four-electron) reduced NOR is capable of two turnovers with NO. Initial binding of NO to reduced heme b(3) occurs with a time constant of approximately 1 micros at 1.5 mM NO, in agreement with earlier studies. This reaction is [NO]-dependent, ruling out an obligatory binding of NO to Fe(B) before ligation to heme b(3). Oxidation of hemes b and c occurs in a biphasic reaction with rate constants of 50 s(-1) and 3 s(-1) at 1.5 mM NO and pH 7.5. Interestingly, this oxidation is accelerated as [NO] is lowered; the rate constants are 120 s(-1) and 12 s(-1) at 75 microM NO. Protons are taken up from solution concomitantly with oxidation of the low spin hemes, leading to an acceleration at low pH. This effect is, however, counteracted by a larger degree of substrate inhibition at low pH. Our data thus show that substrate inhibition in NOR, previously observed during multiple turnovers, already occurs during a single oxidative cycle. Thus, NO must bind to its inhibitory site before electrons redistribute to the active site. The further implications of our data for the mechanism of NO reduction by NOR are discussed.

FIGURE 1.

Absorbance changes during the reaction between fully reduced NOR and NO (1.5 mm). Traces are shown for 430 nm (A, reporting mainly on the heme b₃), 420 nm (B, all hemes contributing), 550 nm (C, heme c) and 560 nm (D, heme b). The initial rise in absorbance at t = 0 at 430 nm is due to the unresolved dissociation of CO. Experimental conditions: 200 mm Hepes at pH 7.5, 50 mm KCl, 0.1 mm EDTA, 0.05% DDM, ∼2 μm reacting NOR, [NO] = 1.5 mm, T = 295 K. The short time scales in A and B were recorded using a brighter light pulse (see “Experimental Procedures”). The red line in the left, shorter panel for 430 nm is a fit to the two time constants 1 μs and 20 μs (see “Experimental Procedures”). The red lines on the longer time scales are two-exponential fits to the two rate constants 50 s⁻¹ and 3 s⁻¹ for oxidation of hemes c and b.

FIGURE 2.

Comparison of the reaction between fully reduced NOR and NO at high (1.5 mm) and lower (75 μm) NO concentrations. Other conditions are as in Fig. 1. The amplitudes of the signals have been normalized to the same CO step. For the traces on the short time scale, a linear slope originating from the pulsed lamp has been subtracted.

FIGURE 3.

Comparison of the oxidation of fully reduced NOR by NO at pH 7.5 and 6.0. Shown are also the differences at high (1.5 mm, A) and low (75 μm, B) [NO]. Other conditions are as in Fig. 1.

FIGURE 4.

A, proton uptake from solution, measured as the change in absorbance of phenol red at 567 nm, during the reaction between fully reduced NOR and NO (75 μm). B, absorbance changes at 430 nm in the same experiment as in A. Experimental conditions: 100 mm KCl (50 mm Hepes/50 mm KCl for the buffered trace), 40 μm phenol red, 50 μm EDTA, 0.05% DDM, pH ∼7.5 (see “Experimental Procedures”). A laser artifact around t = 0 at 567 nm has been truncated for clarity. Other conditions are as in Fig. 1.

FIGURE 5.

Schematic simplified illustration of the reaction between fully reduced NOR and NO. All redox-active cofactors are shown as filled circles when reduced and open circles when oxidized. The reaction starts with the fully reduced NOR after CO is flashed off from heme b₃ (A). NO can now bind, and the b₃-NO intermediate (B) is observed in the kinetic difference spectrum of the rapid (microsecond) phase. The second NO molecule is assumed to bind to Fe_B, although our data are equally consistent with consecutive binding to the NO intermediate at heme b₃. The first turnover is assumed to use electrons from the binuclear site and protons from donors inside the NOR (leading to intermediate C) because no protons are taken from solution and no oxidation of the low spin hemes is observed. Before the second turnover involving oxidation of the low spin hemes, NO must bind to its inhibitory site (D) because we observe inhibition by NO on the rate constant of low spin heme oxidation. Here, we have indicated that this site is the oxidized heme b₃, although our data are equally consistent with Fe_B as the site of inhibition (see “Discussion”). Furthermore, NO binding presumably competes with reformation of the μ-oxo bridge (not shown) between ferric heme b₃ and Fe_B after the first oxidation of the active site. We then observe oxidation of the low spin hemes concomitant with the uptake of protons from the external solution, presumably completing the second turnover, and producing the fully oxidized NOR (E).