Binding of imidazole to the heme of cytochrome c1 and inhibition of the bc1 complex from Rhodobacter sphaeroides: I. Equilibrium and modeling studies

Abstract

We have used imidazole (Im) and N-methylimidazole (MeIm) as probes of the heme-binding cavity of membrane-bound cytochrome (cyt) c(1) in detergent-solubilized bc(1) complex from Rhodobacter sphaeroides. Imidazole binding to cyt c(1) substantially lowers the midpoint potential of the heme and fully inhibits bc(1) complex activity. Temperature dependences showed that binding of Im (K(d) approximately 330 microM, 25 degrees C, pH 8) is enthalpically driven (DeltaH(0) = -56 kJ/mol, DeltaS(0) = -121 J/mol/K), whereas binding of MeIm is 30 times weaker (K(d) approximately 9.3 mM) and is entropically driven (DeltaH(0) = 47 kJ/mol, DeltaS(0)(o) = 197 J/mol/K). The large enthalpic and entropic contributions suggest significant structural and solvation changes in cyt c(1) triggered by ligand binding. Comparison of these results with those obtained previously for soluble cyts c and c(2) suggested that Im binding to cyt c(1) is assisted by formation of hydrogen bonds within the heme cleft. This was strongly supported by molecular dynamics simulations of Im adducts of cyts c, c(2), and c(1), which showed hydrogen bonds formed between the N(delta)H of Im and the cyt c(1) protein, or with a water molecule sequestered with the ligand in the heme cleft.

FIGURE 1.

Spectral changes induced by imidazole binding to ferricyanide-oxidized (solid lines) and ascorbate-reduced (dotted line) bc₁ complex from R. sphaeroides at 25 °C. Inset, absorption changes around λ = 700 nm. Conditions were as follows. 1.5 μm bc₁ in 50 mm Tris, pH 8, 100 mm NaCl, 20 mm cholate, 100 μm ferricyanide, was mixed with 1 mm imidazole (final concentration), and spectra were taken at 15, 30, 60, and 300 s after mixing. Interaction of Im with the ascorbate-reduced bc₁ complex was measured similarly, except the buffer contained 1 mm ascorbate and 10 μm DAD in place of 100 μm ferricyanide. All difference spectra obtained in the presence of ascorbate were virtually identical, and only the last one is shown, after 5 min of incubation with imidazole.

FIGURE 2.

The effect of imidazole on the reducibility of cyt c₁ in bc₁ complex. Cyt bc₁ stock (∼20 μm) in 50 mm Tris, pH 8.0, 100 mm NaCl, 0.01% DM plus 100 μm ferricyanide to fully oxidize the complex were incubated on ice for 5 h without (A) or with (B) 50 mm imidazole. Samples were then diluted 20-fold, and oxidized spectra were recorded. 1 mm ascorbate, 10 μm DAD was then added, and spectra were taken for up to 1 min to ensure that equilibrium was reached (solid lines). The samples were then reduced with a few crystals of sodium dithionite, and fully reduced spectra were obtained after 20–30 s (dashed lines).

FIGURE 3.

Imidazole binding to oxidized cytochrome c₁ at 25 °C. A, spectral changes induced by the addition of Im. B, normalized amplitude of ΔA versus [Im] (from exponential fits to kinetic data, as in A). Dotted line, fit with apparent dissociation constant, K_d = 0.33 mm total imidazole. 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 4.

Binding of imidazole to oxidized cytochrome c₁ of bc₁ complex at 0 °C. A, kinetics of reduction of cyt c₁ by ascorbate after a 24-h incubation on ice with concentrations of imidazole as indicated. B, fraction of high potential, ascorbate-reducible cyt c₁ versus time of incubation on ice with different concentrations of imidazole. At the lowest concentrations of imidazole (≤10 μm), equilibration was not complete even after 250-h incubation at 0 °C, and all data sets were fitted with single exponential functions (dotted lines) (the left vertical axis applies to both A and B). C, equilibrium values of unbound (ascorbate-reducible) cyt c₁ (at t = ∞, from fits in B). Dotted line, fit with apparent dissociation constant, K_d = 40 μm total imidazole. Experimental conditions were as follows. Enzyme stock, containing ∼20 μm bc₁, 50 mm Tris, pH 8.0, 100 mm NaCl, 0.01% DM, 100 μm ferricyanide, was incubated on ice with various concentrations of imidazole and diluted 20-fold with the same buffer except ferricyanide was replaced with 1 mm ascorbate and at room temperature. The fraction of free cyt c₁ was calculated as the ratio of the amount of cyt c₁ reduced by ascorbate after 15 s in samples treated with imidazole versus control without imidazole.

FIGURE 5.

Binding of N-methylimidazole to cytochrome c₁ of R. sphaeroides. A, kinetics of MeIm binding to cyt c₁ at 25 °C (stopped flow traces). B, fraction of cytochrome with MeIm bound versus ligand concentration at 25 °C. Line, fit with apparent dissociation constant K_d = 9.3 mm of total imidazole. Conditions were as follows. 0.5–1 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 6.

The inhibition of bc₁ complex activity (cyt c reduction) by imidazole. A, activity of bc₁ complex, control (upper trace) and preincubated with Im (lower trace). The reaction was started by mixing 2 μl of 20 mm decylubiquinol with 1 ml of buffer (50 mm Tris, pH 8, 100 mm NaCl, 0.01% DM) containing 40 μm horse heart ferricytochrome c and 25 nm bc₁. Preincubation was at 200-fold higher stock concentration with 10 mm imidazole for 2 h on ice. B, relative activity of the bc₁ complex as a function of the c₁-Im fraction obtained from curves similar to those presented in A, except samples were incubated with different Im concentrations. Error bars, S.E. from kinetic fits.

FIGURE 7.

Left, the heme-ligating methionine and adjacent residues in cytochrome c₂ of R. sphaeroides without (black) and with (magenta) imidazole bound (imidazole not shown). In the imidazole-bound state, the “hinge,” located between two long anchoring α-helices (Lime cylinders), has undergone a conformational change coupled to the methionine movement, making room in the heme pocket for small ligand binding (Protein Data Bank entries 1CXA and 1CXC (13)). Right, the structure of the “hinge” region of cytochrome c₁ from R. sphaeroides. The heme is ligated by Met¹⁸⁵ (shown in black). The two long α-helices (Lime cylinders) closest to Met¹⁸⁵ are separated by 68 amino acids. The disulfide bond between Cys¹⁴⁵ and Cys¹⁶⁹ (shown in blue) is present only in cytochrome c₁ from Rhodobacter and is essential to maintain the physiological high potential (Protein Data Bank entry 2FYN (44)). The figure was prepared with VMD (69).

FIGURE 8.

Snapshot from a molecular dynamics simulation of cyt c₁ with imidazole initially in the 180° orientation. Here, a water molecule was trapped near imidazole and remained in approximately the same position over the entire simulation. In most frames, it simultaneously formed hydrogen bonds with the N_δ atom of imidazole and the carbonyl oxygens of Met¹⁸⁵ and Pro¹⁸⁶. This binding mode is very similar to the binding configuration of imidazole to cyt c₂ seen in the 1CXA structure and maintained in the 0° simulation for cyt c₂. The figure was prepared in VMD (69).