Interactions between cytochrome c2 and the photosynthetic reaction center from Rhodobacter sphaeroides: the cation-pi interaction.

Non-covalent molecular interactions play an important role in molecular association processes in biological systems. (1–3) Among these interactions are hydrogen bonds, hydrophobic interactions, and ion-paired salt bridges. (2,3). In addition to these, the cation-pi interaction, the short range electrostatic interaction between a positively charged cation and pi electrons in an aromatic group, (4) has been found to play a role in biological systems. The cation-pi interaction is important in ligand binding (5), in protein structures (6) and in protein-protein complexes (7). This work concerns a cation-pi interaction found in the interface between the cytochrome c2 (cyt c2) and reaction center (RC) of Rhodobacter sphaeroides that is formed between Arg-C32 on the cyt c2 and Tyr-M295 on the RC (8). The importance of this cation –pi interaction on the binding and electron transfer reactions between the two proteins was studied by site directed mutation of both the Arg-C32 on the cyt c2 and Tyr-M295 on the RC. The RC (9,10) is a membrane bound pigment-protein complex in photosynthetic bacteria that performs the initial light-induced electron transfer reactions to convert sunlight into chemical energy (11). Light absorbed by the RC induces electron transfer from a special bacteriochlorophyll dimer, D, the primary donor through series of bound electron acceptors to a bound ubiquinone (QB). The photo-oxidized donor (D+) is reduced by electron transfer from a water soluble cyt c2 allowing flow of electrons through a membrane associated electron transfer chain in a cycle that is coupled to proton pumping that drives ATP synthesis. Operation of the photocycle relies on efficient reactions between the mobile cyt c2 and the membrane-bound RC. For efficient operation, cyt c2 must associate with the RC, transfer electrons and dissociate within the time scale of electron turnover in the cycle (~ 10−3 s) (12). The binding and electron transfer rates of isolated cyt c2 and RC have been extensively studied using laser pulse kinetic measurements (13–18). The reduction of the oxidized donor D+ by reduced cyt c2 shows two kinetic phases following a single laser flash – a fast (μs) first order phase (independent of cyt c2 concentration) due to electron transfer from bound cyt c2 to the photo-oxidized donor of the RC and a slower (ms) second order phase (dependent on cyt c2 concentration) due to the binding and subsequent electron transfer of free cyt c2. The observed biphasic kinetics can be explained by the following scheme (17). cytc22++DQB↔KDcytc22+:DQB↓hvcytc22++D+QB−⇄koffkoncytc22+:D+QB−→kecytc23+:DQB− (1) where KD is the dissociation constant, kon is the association rate constant, koff is the dissociation rate constant and ke is the electron transfer rate constant in the bound state. The equilibrium between bound and free cyt c2 is achieved in the dark. Following a laser flash the re-reduction of D+ by cyt c22+ is biphasic. RCs with a bound cyt c2 undergo rapid electron transfer with a rate constant ke ( 106 s−1) (14). RCs without a bound cyt c2 undergo slower diffusion limited electron transfer with an observed second order rate constant k2 (~109 s−1M−1) (18). Since ke ≫ koff (19), the observed second order rate constant is the association rate, k2 ~ kon. (20) The fraction of RCs with a bound cyt c2 can be determined by the ratio of the fast and slow phases. The issociation constant KD can be determined from a plot of the fraction of RCs with bound cyt c2 versus the free cyt c2 concentration. The importance of electrostatic interactions on binding and electron transfers between cyt c2 and the RC has been established by the ionic strength dependence of k2 (13), the effect of site directed mutation of charged residues (17,21), chemical cross-linking (22–24) and by electrostatic modeling (15,25,26). Hydrophobic interactions, particularly those of Tyr L162 on the RC have been shown to be important for binding and electron transfer. (18, 27). The structure of the cyt c2:RC complex was obtained by co-crystallizing the two proteins in a photochemically active complex and determining its x-ray crystal structure. (8) The structure of the complex shows the binding site to be between the cyt c2 and the periplasmic surface of the RC with a central short range contact region in which the exposed heme edge is in contact with Tyr-L162, directly above the BChl dimer (Figure 1). The close contact between the two proteins provides an efficient tunneling pathway for fast electron transfer in the bound state. In the central region the residues from the two proteins make contact through van der Waals, and hydrogen bonding interactions as well as the cation-pi interaction (Figure 1). The closest distance between the positively charged guanidinium group of Arg-C32 and the phenolic group of Tyr-M295 is less than 4 A, characteristic of a cation-pi complex (6). Surrounding the central region of close contact is a solvent separated region having positively charged residues on the cyt c2 positioned opposite negatively charged residues on the RC interface between the two proteins. The charged residues do not form salt bridges but are separated by solvent. Figure 1 The cation-pi interaction in the RC: cyt c2 complex (PDB accession code 1L9B, ref. 8). The RC subunits L, M, and H are shown in yellow, blue, and green, respectively. The primary donor (bacteriochlorophyll dimer) of the RC is shown in red. The cyt c2 ... In this work, the functional role of the cation-pi interaction in the binding, association and electron transfer between cyt c22+ and the RC is addressed in order to answer the following questions. (1) What is the magnitude of this interaction? (2) Is this interaction important for the association of cyt c2 and the RC? (3) Is it important for inter-protein electron transfer? To answer these questions site directed mutants of the RC at Tyr-M295 and cyt c2 at Arg-C32 were constructed. Mutants were created that replaced the aromatic residue Tyr-M295 on the RC with either aromatic or non-aromatic residues, and replaced the cationic residue Arg-C32 on cyt c2 with either the cationic Lys residue or neutral residues. The effect of these mutations on binding and electron transfer rates were measured by transient absorption spectroscopy using flash photolysis and compared to native values.