Binding of Imidazole to the Heme of Cytochrome c1 and Inhibition of the bc1 Complex from Rhodobacter sphaeroides

Abstract

The kinetics of imidazole (Im) and N-methylimidazole (MeIm) binding to oxidized cytochrome (cyt) c 1 of detergentsolubilized 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 ϳ1 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 ratelimiting 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.The binding of small molecules to c-type hemes has been widely studied in type I cytochromes c and is known to result in displacement of the native methionine ligand and to cause significant conformational changes in the heme binding domain (1-4). The affinities and kinetics of binding reflect the interactions among the exogenous ligand, the heme, and the protein and provide a window into the static and dynamic properties of the heme domain.In cytochromes (cyts) 2 c and c 2 , the methionine ligand of the heme is located in the middle of a 12-18-residue linker connecting two ␣-helices of the conserved cytochrome c fold. Structural studies of cyts c and c 2 with exogenous ligands bound (1, 2, 5) show that part of the linker sequence (termed the "hinge" by Dumortier et al. (3)) is rotated to create a slight opening or loosening of the heme binding cavity. The initial and final states are generally referred to as closed and open. However, there is no direct evidence that ligand binding is preceded by a spontaneous conformation change that closely resembles the open state seen in ligand-bound structures or that the heme-methionine linkage is actually broken in any intermediate state. Indeed, the mechanism of ligand entry and access to the heme is still unknown.Here we report on the kinetics of binding of imidazole and N-methylimidazole to cytochrome c 1 in isolated cyt bc 1 complex from Rhodobacter sphaeroides. Cytochrome c 1 is an integral component of the bc 1 complex, playing the role of electron carrier between the iro...

Isolation and biophysical characterization of GSU0105, a triheme c-type cytochrome fromGeobacter sulfurreducens

“…This is consistent with globular monomeric protein with a size comparable to PpcA [41]. Displacement of native axial methionine ligands by imidazole in metal affinity chromatography preparations is a well-established phenomenon [59][60][61]. It is likely that in concentrated GSU0105 solutions, during dialysis or other steps for residual imidazole removal, one of the axial heme ligands in GSU0105 can be replaced by a histidine residue belonging to the His-tag of a different cytochrome molecule instead of properly refolding with the native methionine ligand in place.…”

The acidic domain of cytochrome c1 in Paracoccus denitrificans, analogous to the acidic subunits in eukaryotic bc1 complexes, is not involved in the electron transfer reaction to its native substrate cytochrome c552

“…The reducibility of the cytochrome c 1 heme by ascorbate was strongly diminished, as well as the turnover number (results not shown). A similar effect has been described for complex III of Rhodobacter capsulatus [35], where high imidazole concentrations diminished the activity drastically (20 fold lower than wild-type) and impaired the reducibility of cytochrome c 1 by displacing of the heme liganding Met [36, 37]. Changing the elution buffer from imidazole to histidine kept the redox properties of cytochrome c 1 intact and gave turnover numbers comparable to the wild-type enzyme (see below).…”

Structural Characterization of Neisseria gonorrhoeae Bacterial Peroxidase—Insights into the Catalytic Cycle of Bacterial Peroxidases

“…Analysis of the UV-visible spectra indicated that the E heme methionine S-Fe bond was relatively weak (a temperature-dependent high/low-spin equilibrium was observed [ 5 ]); thus, at concentrations above 50 mM imidazole, it was proposed that imidazole competes and dissociates the methionine from the ferrous heme. This effect has been reported for the binding of exogenous ligands to cytochrome c 1 , in which it was observed that when imidazole was titrated the usually observed hyperbolic dependence on ligand concentration became more complex [ 28 ]. Similar to our proposal for Ng BCCP, the authors explained the result as imidazole promoting a conformational change followed by bond formation with the heme iron [ 28 ].…”