Probing the Role of the Fe−S Subunit Hinge Region during Q_o Site Catalysis in Rhodobacter capsulatus bc₁ Complex

Abstract

The ubihydroquinone:cytochrome c oxidoreductase, or bc(1) complex, functions according to a mechanism known as the modified Q cycle. Recent crystallographic data have revealed that the extrinsic domain containing the [2Fe2S] cluster of the Fe-S subunit of this enzyme occupies different positions in various crystal forms, suggesting that this subunit may move during ubihydroquinone oxidation. As in these structures the hydrophobic membrane anchor of the Fe-S subunit remains at the same position, the movement of the [2Fe2S] cluster domain would require conformational changes of the hinge region linking its membrane anchor to its extrinsic domain. To probe the role of the hinge region, Rhodobacter capsulatus bc(1) complex was used as a model, and various mutations altering the hinge region amino acid sequence, length, and flexibility were obtained. The effects of these modifications on the bc(1) complex function and assembly were investigated in detail. These studies demonstrated that the nature of the amino acid residues located in the hinge region (positions 43-49) of R. capsulatus Fe-S subunit was not essential per se for the function of the bc(1) complex. Mutants with a shorter hinge (up to five amino acid residues deletion) yielded functional bc(1) complexes, but contained substoichiometric amounts of the Fe-S subunit. Moreover, mutants with increased rigidity or flexibility of the hinge region altered both the function and the assembly or the steady-state stability of the bc(1) complex. In particular, the extrinsic domain of the Fe-S subunit of a mutant containing six proline residues in the hinge region was shown to be locked in a position similar to that seen in the presence of stigmatellin. Interestingly, the latter mutant readily overcomes this functional defect by accumulating an additional mutation which shortens the length of the hinge. These findings indicate that the hinge region of the Fe-S subunit of bacterial bc(1) complexes has a remarkable structural plasticity.

Binding Dynamics at the Quinone Reduction (Qi) Site Influence the Equilibrium Interactions of the Iron Sulfur Protein and Hydroquinone Oxidation (Qo) Site of the Cytochromebc1Complex

“…All R. capsulatus strains were grown in mineral-peptone-yeast-extract enriched media (MPYE) under semi-aerobic conditions in the dark at 35° C, as described previously (10). The construction and growth phenotypes of the H212N and H217L, D and R mutants have been described previously in (8,10,22), respectively.…”

“…The construction and growth phenotypes of the H212N and H217L, D and R mutants have been described previously in (8,10,22), respectively.…”

“…For example, why in the presence of antimycin A, electrons deposited to the heme b H following a QH 2 oxidation at the Q o site do not leak back to an oxidized cyt c 1 via the natural oscillation of the Fe/S protein (13-15). Similarly, it is not obvious why the macro-movement of the extrinsic domain of the Fe/S protein, during which it interacts closely with the solvent exposed cd 1 , cd 2 and ef loops of cyt b (10,16,17), is required for multiple turnovers of the cyt bc 1 . These and other observations often raise the issue of whether there exists in the cyt bc 1 some mechanism(s) preventing the occurrence of short circuits, especially during the onset of subsequent Q o site turnovers.…”

Functional Insensitivity of the Cytochrome b6f Complex to Structure Changes in the Hinge Region of the Rieske Iron-Sulfur Protein

“…that introduction of excess flexibility by substitution with 6 Gly residues (19) or of extra length by insertion of 1 or more residues (17,24,26) in the hinge region is deleterious to the function of the bc 1 complex (Table III, parts A and B). With 4 -7 glycine residues in the sequence, the ISP hinge region in the b 6 f complex is predicted to be structure-less and more flexible than that in the bc 1 complex (Table I).…”

“…It was subsequently found that the function of the complex is very sensitive to perturbation of the sequence and structure of the hinge region by sitedirected mutagenesis in which the hinge was altered by residue deletion, insertion, and substitution (17)(18)(19)(20)(21)(22)(23)(24)(25)(26). Although these data were interpreted in terms of a requirement for the mobility of the ISP soluble domain, the extreme sensitivity of bc 1 function to increases in hinge flexibility (19) and length (17,24,26) cannot be predicted or inferred from the x-ray structure data. Thus, it is of interest to study the function of the putative hinge region in the b 6 f complex (Table I).…”