Uncovering the [2Fe2S] domain movement in cytochrome bc ₁ and its implications for energy conversion

Abstract

In crystals of the key respiratory and photosynthetic electron transfer protein called ubihydroquinone:cytochrome (cyt) c oxidoreductase or cyt bc 1, the extrinsic [2Fe2S] cluster domain of its Fe-S subunit assumes several conformations, suggesting that it may move during catalysis. Herein, using Rhodobacter capsulatus mutants that have modifications in the hinge region of this subunit, we were able to reveal this motion kinetically. Thus, the bc1 complex (and possibly the homologous b6f complex in chloroplasts) employs the [2Fe2S] cluster domain as a device to shuttle electrons from ubihydroquinone to cyt c1 (or cyt f ). We demonstrate that this domain movement is essential for cyt bc 1 function, because a mutant enzyme with a nonmoving Fe-S subunit has no catalytic activity, and one with a slower movement has lower activity. This motion is apparently designed with a natural frequency slow enough to assure productive Qo site charge separation but fast enough not to be rate limiting. These findings add the unprecedented function of intracomplex electron shuttling to large-scale domain motions in proteins and may well provide a target for cyt bc 1 antibiotics.Rhodobacter capsulatus ͉ photosynthetic and respiratory electron transfer ͉ mitochondrial complex III ͉ protein domain motion ͉ Rieske Fe-S subunit W hen different crystal structures reveal dramatically different protein conformations, large amplitude domain movements are often inferred. However, in only a few cases such as myosin (1), flagellar motor (2), and ATP synthase (3, 4) have such movements been visualized. The cytochrome (cyt) bc 1 (or its cyt b 6 f counterpart in chloroplasts) is a key component of respiratory and photosynthetic electron transfer chains (5, 6). Recent crystal structures of the mitochondrial cyt bc 1 have revealed that the extrinsic [2Fe2S] cluster domain of the Fe-S subunit occupies various locations within this enzyme complex (7-10). It has been observed in either a position proximal to the ubihydroquinone (QH 2 ) oxidation catalytic site (Q o position) from which it takes electrons or a position close to cyt c 1 subunit (c 1 position) to which it donates electrons (Fig. 1). Because of the large distances observed between the electron-donating and electron-accepting cofactors of the cyt bc 1 in the different structures, no one of these locations can support sufficiently rapid electron tunneling (11) to meet the observed turnover rates (12, 13) and the specific substrate-product interactions (14) that occur at the QH 2 oxidation site. Thus, an unprecedented intracomplex electron shuttle motion to transfer electrons during catalysis has been suggested (8). However, neither the presumably essential movement nor the electron transfer associated with it has been visualized before this work.In light-activated energy transduction systems, such as the one provided by the photosynthetic bacterium R. capsulatus, a short flash of light (Ͻ10-s duration) can activate the photochemical reaction center, thereby inducing oxidation of two equival...

X-Ray Structure of Rhodobacter Capsulatus Cytochrome bc1: Comparison with its Mitochondrial and Chloroplast Counterparts

“…This strain overproduces the cyt bc 1 by about five to eight fold when grown in enriched medium under semi-aerobic conditions. Intracytoplasmic membranes (chromatophores) were obtained by differential centrifugation after breaking the cells using a French press, as described earlier (Darrouzet, Valkova-Valchanova et al 2000) The cyt bc 1 was isolated by ion-exchange chromatography of a dodecyl maltoside extract, essentially as described (Berry et al 1991). Specifically, chromatophore membranes were solubilized at 10 g/l protein in 50 mM potassium phosphate buffer (KPi) pH 7.3 containing 260 mM NaCl, 0.1 mM PMSF (added freshly from an ethanolic solution) and dodecyl-β-D-maltoside (DM) at a detergent:protein ratio of 1:1.…”

“…The same crystal form has been obtained without stigmatellin, however the resolution (7 Å) was not high enough to describe the occupation of the Qo site. Efforts are currently under way to crystallize one of the "neck" mutants (Darrouzet et al 2000;Cooley et al 2004), which seem to hold the ISP prefferentially in a position similar to the stigmatellin position, in the absence of stigmatellin.…”

Movement of the Iron−Sulfur Head Domain of Cytochrome bc1 Transiently Opens the Catalytic Qo Site for Reaction with Oxygen

“…Those properties knock out the physiological competence of the enzyme by introducing either a thermodynamic or a steric barrier for one of the steps that follows the initial oxidation of quinol at the Q o site. In the FeS motion knockout, the FeS head domain is sterically arrested close to the Q o site, which severely interferes with its normal oscillation between the Q o site and heme c 1 required for efficient electron transfer (27). In the c 1 knockout, the midpoint potential of heme c 1 is lowered by more than 300 mV, making the electron transfer through this heme extremely uphill (26).…”

“…Mutated cytochrome bc 1 included the following cofactor knockout forms: the c 1 knockout with the M183L mutation in cytochrome c 1 (26) and the FeS motion knockout with the +2Ala insertion in the neck region of the FeS subunit (27 enzymatic activity of cytochrome bc 1 was assayed by measuring the DBH 2 (2,3-dimethoxy-5-decyl-6-methyl-1,4-benzohydroquinone)-dependent reduction of mitochondrial horse cytochrome c as described previously (25). The measurements of superoxide production, based on superoxide dismutase-sensitive reduction of cytochrome c, were performed as described previously (5).…”

“…Wild-type and mutated cytochrome bc 1 were isolated from the appropriate strains (25)(26)(27) of Rhodobacter capsulatus as described previously (28). Mutated cytochrome bc 1 included the following cofactor knockout forms: the c 1 knockout with the M183L mutation in cytochrome c 1 (26) and the FeS motion knockout with the +2Ala insertion in the neck region of the FeS subunit (27 enzymatic activity of cytochrome bc 1 was assayed by measuring the DBH 2 (2,3-dimethoxy-5-decyl-6-methyl-1,4-benzohydroquinone)-dependent reduction of mitochondrial horse cytochrome c as described previously (25).…”