Double Mutant Studies Identify Electrostatic Interactions That Are Important for Docking Cytochrome c₂ onto the Bacterial Reaction Center

Abstract

Cytochrome c2 (cyt) is the mobile electron donor to the reaction center (RC) in photosynthetic bacteria. The electrostatic interactions involved in the dynamics of docking of cyt onto the RC were examined by double mutant studies of the rates of electron transfer between six modified Rhodobacter sphaeroides RCs in which negatively charged acid residues were replaced with Lys and five modified Rhodobacter capsulatus Cyt c2 molecules in which positively charged Lys residues were replaced with Glu. We measured the second-order rate constant, k2, for electron transfer from the reduced cyt to the oxidized primary donor on the RC, which reflects the energy of the transition state for the formation of the active electron transfer complex. Strong interactions were found between Lys C99 and Asp M184/Glu M95, and between Lys C54 and Asp L261/Asp L257. The interacting residues were found to be located close to each other in the recently determined crystal structure of the cyt-RC complex [Axelrod, H., et al. (2002) J. Mol. Biol. (in press)]. The interaction energies were approximately inversely proportional to the distances between charges. These results support earlier suggestions [Tetreault, M., et al. (2001) Biochemistry 40, 8452-8462] that the structure of the transition state in solution resembles the structure of the cyt-RC complex in the cocrystal and indicate that specific electrostatic interactions facilitate docking of the cyt onto the RC in a configuration optimized for both binding and electron transfer. The specific interaction between Asp M184 and Lys C99 may help to nucleate short-range hydrophobic contacts.

Transition state and encounter complex for fast association of cytochrome c 2 with bacterial reaction center

“…The positions of some of these critical residues in the EC and TS are shown in Fig. 6 b and c. These residues are found on the Cyt surface near C99, C93, and C54 in the center of the positively charged region of the Cyt that interacts with the center of the negatively charged cluster of residues centered around M184, M95, and M292 (25). These interactions help to guide the Cyt to its position in the bound active state.…”

“…To determine the structure of the TS, we used the experimentally determined changes in TS free energies due to mutation of charged groups, ␦⌬G exp ‡ (25), and compare them with the calculated changes in electrostatic energy for the Cyt͞RC complex arranged at different docking positions. We assume that the TS ensemble for the binding process consists of a small set of closely related structures that does not change greatly by mutation, and that the change in free energy of the TS, ␦⌬G ‡ is determined only by the electrostatic interactions between mutated charged residues.…”

“…Changes to charge-surface groups near the binding interface by using chemical modification (22,23) and site-directed mutagenesis (13,24,25) were shown to change the association rate. The role of electrostatic interactions in protein association was demonstrated by modeling studies (26)(27)(28)(29)(30).…”

“…capsulatus Cyt with Rb. sphaeroides RCs are very similar (25). The second-order reaction rates were measured for a series of mutant RCs and Cyts having charge-reversing mutations to charged residues, six modified RCs with acid 3 Lys changes, and five modified Cyts with Lys 3 Glu changes, a total of 42 reaction combinations.…”

Surface Charges and Regulation of FMN to Heme Electron Transfer in Nitric-oxide Synthase

“…Previous work identified some protein structural features that may influence this step in NOS enzymes (17,19,20,30,42,44). The electron transfer may rely on complementary electrostatic interaction between charged surface residues (18,19), as described for flavodoxins, dual-flavin enzymes, and other enzyme systems (32,(45)(46)(47)(48)(49). This work is the first to examine the individual roles of eight residues that create a positive charge cluster on the NOSoxy domain.…”