Role of charges on cytochrome f from the cyanobacterium Phormidium laminosum in its interaction with plastocyanin.
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.
Hart
Sarah E
SE
Department of Biochemistry and Cambridge Centre for Molecular Recognition, University of Cambridge, Cambridge, CB2 1QW, United Kingdom.
Schlarb-Ridley
Beatrix G
BG
Delon
Christine
C
Bendall
Derek S
DS
Howe
Christopher J
CJ
D019943:Amino Acid Substitution; D001665:Binding Sites; D000458:Cyanobacteria / Q000378:metabolism*; D003580:Cytochromes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D045348:Cytochromes f; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009994:Osmolar Concentration; D010970:Plastocyanin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation
D003580:Cytochromes; D010970:Plastocyanin; D045348:Cytochromes f