Binding of imidazole to the heme of cytochrome c1 and inhibition of the bc1 complex from Rhodobacter sphaeroides: II. Kinetics and mechanism of binding

Abstract

The kinetics of imidazole (Im) and N-methylimidazole (MeIm) binding to oxidized cytochrome (cyt) c(1) of detergent-solubilized bc(1) complex from Rhodobacter sphaeroides are described. The rate of formation of the cyt c(1)-Im complex exhibited three separated regions of dependence on the concentration of imidazole: (i) below 8 mM Im, the rate increased with concentration in a parabolic manner; (ii) above 20 mM, the rate leveled off, indicating a rate-limiting conformational step with lifetime approximately 1 s; and (iii) at Im concentrations above 100 mM, the rate substantially increased again, also parabolically. In contrast, binding of MeIm followed a simple hyperbolic concentration dependence. The temperature dependences of the binding and release kinetics of Im and MeIm were also measured and revealed very large activation parameters for all reactions. The complex concentration dependence of the Im binding rate is not consistent with the popular model for soluble c-type cytochromes in which exogenous ligand binding is preceded by spontaneous opening of the heme cleft, which becomes rate-limiting at high ligand concentrations. Instead, binding of ligand to the heme is explained by a model in which an initial and superficial binding facilitates access to the heme by disruption of hydrogen-bonded structures in the heme domain. For imidazole, two separate pathways of heme access are indicated by the distinct kinetics at low and high concentration. The structural basis for ligand entry to the heme cleft is discussed.

FIGURE 1.

Rate of imidazole binding to oxidized cytochrome c₁ in the low concentration range (≤50 mm). Solid line, hyperbolic fit to data points in the range 10–50 mm imidazole: V_max = 1.1 s⁻¹, K_m = 15 mm. Dashed line, linear fit to data points in the range 0.1–2 mm: slope = 27 m⁻¹ s⁻¹. Conditions were as follows. 1 μm oxidized bc₁ complex in 50 mm Tris, pH 8.0, 100 mm NaCl, 20 mm cholate was mixed with various amounts of Im at 25 °C.

FIGURE 2.

Imidazole and N-methylimidazole binding to oxidized cytochrome c₁. Top, MeIm in 100 mm NaCl. Solid line, hyperbolic fit to all data for [MeIm] < 800 mm, with V_max = 70 s⁻¹, K_m = 1000 mm, k_off = 1.4 s⁻¹. Bottom, closed circles, Im in 100 mm NaCl; open circles, Im in 900 mm NaCl. Solid line, fit according to Scheme 4 with parameters as given in Table 3. Conditions were as follows. 1 μm oxidized bc₁ complex in 50 mm Tris, pH 8.0, 100 or 900 mm NaCl, 20 mm cholate was mixed with various amounts of Im or MeIm at 25 °C.

FIGURE 3.

Rate of imidazole and N-methylimidazole release from cytochrome c₁. A, reduction of cyt c₁ upon release of Im and MeIm, at 25 °C. (The MeIm kinetics are readily resolved on a faster time scale.) The apparent release rate constant, k_obs(off), is 0.012 s⁻¹ for Im and 0.79 s⁻¹ for MeIm. B, temperature dependence of the release rate of Im (circles) and MeIm (squares). Conditions were as follows. Oxidized bc₁ stock (20–50 μm) was preincubated with 10 mm Im or MeIm for at least 2 h, on ice, diluted to a final concentration of 0.5 μm bc₁ in 50 mm Tris, pH 8.0, 100 mm NaCl, 20 mm cholate, and immediately mixed with ascorbate and DAD (final concentrations 1 mm and 10 μm, respectively).

FIGURE 4.

Impact of imidazole binding on the heme binding cleft of cytochrome c₁. Left images, no imidazole (from Protein Data Bank entry 2FYN). Right images, imidazole bound (from MD simulation). Top, view along the exposed heme ring D-C edge. Bottom, view from above. Residues shown are Ala¹⁸⁴–Pro¹⁸⁸. The figure was prepared with VMD (42).