Role of Charges on Cytochrome f from the Cyanobacterium Phormidium laminosum in Its Interaction with Plastocyanin

• Published in: Biochemistry (Easton) 2003-05, Vol.42 (17), p.4829-4836
• Main Authors: Hart, Sarah E, Schlarb-Ridley, Beatrix G, Delon, Christine, Bendall, Derek S, Howe, Christopher J
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution; Binding Sites; Cyanobacteria - metabolism; Cyanophyta; cytochrome f; Cytochromes - chemistry; Cytochromes - genetics; Cytochromes - metabolism; Cytochromes f; Kinetics; Models, Molecular; Mutagenesis, Site-Directed; Osmolar Concentration; Phormidium laminosum; plastocyanin; Plastocyanin - chemistry; Plastocyanin - genetics; Plastocyanin - metabolism; Protein Conformation
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi020674h

The role of charge on the surface of cytochrome f from the cyanobacterium Phormidium laminosum in the reaction with plastocyanin was investigated in vitro using site-directed mutagenesis. Charge was neutralized at five acidic residues individually and introduced at a residue close to the interface between the two proteins. The effects on the kinetics of the reaction were measured using stopped-flow spectrophotometry, and the midpoint potentials of the mutant proteins were determined. The dependence of the bimolecular rate constant of reaction, k 2, on ionic strength was determined for the reactions of the cytochrome f mutants with wild-type and mutant forms of plastocyanin. Double mutant cycle analysis was carried out to probe for the presence of specific electrostatic interactions. The effects of mutations on Cyt f were smaller than those seen previously for mutants of plastocyanin [Schlarb-Ridley, B. G. et al. (2002) Biochemistry 41, 3279−3285]. One specific short-range interaction between charged residues of wild-type plastocyanin (Arg93) and wild-type cytochrome f (Asp63) was identified. The kinetic evidence from this study and that of Schlarb-Ridley et al., 2002, appears to conflict with the NMR structure of the P. laminosum complex, which suggests the absence of electrostatic interactions in the final complex [Crowley, P. et al. (2001) J. Am. Chem. Soc. 123, 10444−10453]. The most likely explanation of the apparent paradox is that the overall rate is diffusion controlled and that electrostatics specifically influence the encounter complex and not the reaction complex.