title: Interaction between cytochrome c2 and the photosynthetic reaction center from Rhodobacter sphaeroides: effects of charge-modifying mutations on binding and electron transfer.
abstract: The electrostatic interactions governing binding and electron transfer from cytochrome c(2) (cyt c(2)) to the reaction center (RC) from the photosynthetic bacteria Rhodobacter sphaeroides were studied by using site-directed mutagenesis to change the charges of residues on the RC surface. Charge-reversing mutations (acid --> Lys) decreased the binding affinity for cyt c(2). Dissociation constants, K(D) (0.3--250 microM), were largest for mutations of Asp M184 and nearby acid residues, identifying the main region for electrostatic interaction with cyt c(2). The second-order rate constants, k(2) (1--17 x 10(8) M(-1) s(-1)), increased with increasing binding affinity (log k(2) vs log 1/K(D) had a slope of approximately 0.4), indicating a transition state structurally related to the final complex. In contrast, first-order electron transfer rates, k(e), for the bound cyt did not change significantly (<3-fold), indicating that electron tunneling pathways were unchanged by mutation. Charge-neutralizing mutations (acid --> amide) showed changes in binding free energies of approximately 1/2 the free energy changes due to the corresponding charge-reversing mutations, suggesting that the charges in the docked complex remain well solvated. Charge-enhancing mutations (amide --> acid) produced free energy changes of the same magnitude (but opposite sign) as changes due to the charge-neutralizing mutations in the same region, indicating a diffuse electrostatic potential due to cyt c(2). A two-domain model is proposed, consisting of an electrostatic docking domain with charged surfaces separated by a water layer and a hydrophobic tunneling domain with atomic contacts that provide an efficient pathway for electron transfer.
authors.affiliation: Physics Department, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093, USA.
authors.forename: M
authors.initials: M
authors.lastname: Tetreault
authors.forename: S H
authors.initials: SH
authors.lastname: Rongey
authors.forename: G
authors.initials: G
authors.lastname: Feher
authors.forename: M Y
authors.initials: MY
authors.lastname: Okamura
mesh_terms: D003574:Cytochrome c Group / Q000737:chemistry / Q000378:metabolism*; D045362:Cytochromes c2; D004579:Electron Transport / Q000235:genetics; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010782:Photolysis; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D012242:Rhodobacter sphaeroides / Q000201:enzymology* / Q000235:genetics; D055672:Static Electricity; D013816:Thermodynamics
chemical_list: D003574:Cytochrome c Group; D046911:Macromolecular Substances; D045322:Photosynthetic Reaction Center Complex Proteins; D045362:Cytochromes c2