Plasmon waveguide resonance spectroscopic evidence for differential binding of oxidized and reduced Rhodobacter capsulatus cytochrome c2 to the cytochrome bc1 complex mediated by the conformation of the Rieske iron-sulfur protein.

In its simplest form, bacterial photosynthesis involves electron transfer between two membrane-bound complexes, the photosynthetic reaction center (RC) and cytochrome bc1 (BC1), which is mediated by lipid-soluble quinone and by water-soluble cytochrome c2 (C2). Upon absorption of light, the special pair bacteriochlorophyll becomes oxidized and the quinone (QB) reduced. The reaction center is rapidly reduced by C2 to complete the initial steps of the reaction. In the second half of the reaction, quinol is oxidized by the cytochrome b (B) and the Rieske iron-sulfur protein (ISP). The Rieske ISP transfers its electron to cytochrome c1 (C1), which in turn reduces C2 to complete the cycle. During reduction of quinone, protons are taken up from the cytoplasmic side of the membrane; during subsequent oxidation of quinol, the protons are released on the periplasmic side, resulting in a proton gradient that is used to synthesize ATP. The three-dimensional structures of RC (1, 2), BC1 (3-6), and C2 (7, 8) have been determined. In addition, the crystal structures of the complexes of C2 and RC (9) and of cytochrome c (C) and BC1 (10) are known. Thus, the structures of all of the components of the pathway and their immediate interaction partners have been determined, but what is missing is the precise mechanism by which the electron transfer process is carried out and turnover optimized. To date, considerable attention has been focused on the role of C2 in mediating electron transfer between reduced BC1 and the oxidized RC. Studies of the kinetics of electron transfer have emphasized the contribution of electrostatic interactions between negative charges on RC and positive charges on C2 to facilitate binding and electron transfer at low ionic strength. However, hydrophobic interactions and hydrogen bonding also contribute to binding. Thus, it has been proposed that electrostatics steer the C2 toward the optimum binding site on the RC, but that hydrophobic interactions then provide the primary stabilization of the bound complex (11, 12). Binding constants typically have been indirectly derived from kinetics of electron transfer (e.g. 13), although a few direct measurements have been performed (e.g. 14). It is well established that C2, and more generally Class I c-type cytochromes, are substantially less stable in the oxidized state as compared to the reduced state due to the buried positive charge on the ferric heme iron. This differential stability is in part manifested by a dynamic conformational change that is present in only the oxidized cytochrome. This dynamic process involves the breakage of the methionyl sulfur-Fe+3 bond and the rapid movement (∼ 30 s−1) of the methionine (M96 in Rb. capsulatus C2) and adjacent amino acids (∼ positions 90 – 100, the so-called “hinge region”) away from the heme face (15). We recently found, using plasmon waveguide resonance (PWR) spectroscopy, that dissociation of the oxidized C2 (C2 (ox)) – reduced RC complex is facilitated by the hinge movement (16). Key to these studies was the use of the C2 mutant K93P which has hinge kinetics that are ∼25 fold faster than in the wild-type cytochrome (∼700 s−1) and 30-fold weaker binding to RC. In the case of RCs, reduced wild-type C2 binds to both a high- and low-affinity site, while wild-type C2 (ox) and both redox states of the mutant C2 can only bind to the low-affinity site. This was attributed to the more stable structure of reduced wild-type C2 and to a conformational change occurring upon oxidation of C2, which occurs more readily in the oxidized K93P mutant, resulting in a form incapable of high-affinity binding (16). There have been a number of studies on the interaction of cytochromes (C or C2) with BC1 (reviewed by Millett & Durham, 17). There are strong parallels with the RC studies in that the C2/BC1 interaction has a large electrostatic component that steers the reactants for optimum binding, which is primarily hydrophobic in nature. In the yeast system, intracomplex electron transfer from the Rieske ISP to C1 occurs with a rate constant of ∼80,000 s−1, although the overall reaction is gated by movement of the Rieske ISP between the B and C1 centers (17). Within the preformed complex of C and BC1 at low ionic strength, electron transfer from C1 to C also is very fast, occurring with a rate constant of ∼14,000 s−1, with an apparent μM dissociation constant (18). One might expect a similar mechanism for substrate binding and product release as with RC, i.e. a dynamic conformational change that facilitates product release and thus turnover. The products in the case of reaction with BC1 are reduced C2 (C2 (red)) and oxidized C1 (C1 (ox)), with C2 (red) being a relatively stable structure with no “hinge” movement. However, both C1 and C2 are class I cytochromes that share a common fold. Thus, it is possible that C1 (ox) undergoes a dynamic conformational change to facilitate product release. Note however, that the situation with BC1 is somewhat more complex than RC as the Rieske ISP has been shown to be mobile and can be found proximal to both the B and to the C1 depending on conditions (4). Movement of the Rieske ISP has been very well documented in several species and is an integral part of the mechanism (19-23). Indeed, in the presence of the BC1 inhibitor stigmatellin, the Rieske ISP associates with the B cytochrome and the iron-sulfur cluster is distal from C1 in the BC1 complex. In what follows, we report PWR studies on the interaction of Rhodobacter capsulatus C2 (oxidized and reduced) with BC1 in a phospholipid bilayer, using wild-type and mutant C2 (K93P) and the inhibitor stigmatellin.