Study of the cytochrome c2-reaction center interaction by site-directed mutagenesis.
Photooxidation of Rhodobacter capsulatus cytochrome c2 and four site-directed mutants by detergent solubilized Rhodobacter sphaeroides reaction centers was studied as a function of ionic strength at pH 8.0. Mutants of cytochrome c2 included K12D (lysine 12 substituted by aspartate), K14E (lysine 14 substituted by glutamate), K32E (lysine 32 substituted by glutamate), and K14E/K32E (lysines 14 and 32 substituted by glutamates). With respect to the wild-type, the mutants exhibited decreased second-order rate constants, indicating perturbation of their electrostatic interaction with the reaction center. In the transient complex, the interaction domain charges of the reaction center and wild-type cytochrome c2 were estimated to be -4.8 and +4.8, respectively. In contrast, the interaction domain charges of mutants K12D, K14E, K32E, and K14E/K32E were estimated to be +2.8, +3.7, +3.6 and +1.3, respectively. At infinite ionic strength, the second-order rate constant of the wild-type cytochrome c2 photooxidation (k infinity) was estimated to be 8.7 x 10(6) M-1 s-1. In the case of K32E, k infinity was not changed significantly (8.2 x 10(6) m-1 s-1), suggesting that the electrostatic perturbation of this mutant was largely overcome at high ionic strength. In contrast, the k infinity for K12D, K14E, and K14E/K32E were estimated to be decreased 2-7-fold. Consequently, mutations to R. capsulatus lysines 12 and 14 appear to perturb the distance and/or orientation of the cytochrome c2 relative to the reaction center in the reactive complex, as well as alter electrostatic interactions. Based upon the kinetic results presented here, the cytochrome c2-reaction center transient complex has been modeled.
Caffrey
M S
MS
Department of Biochemistry, University of Arizona, Tucson 85721.
Bartsch
R G
RG
Cusanovich
M A
MA
D000595:Amino Acid Sequence; D001665:Binding Sites; D003574:Cytochrome c Group / Q000235:genetics* / Q000378:metabolism*; D045362:Cytochromes c2; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D010777:Photochemistry; D011487:Protein Conformation; D016354:Rhodobacter capsulatus / Q000378:metabolism*; D012242:Rhodobacter sphaeroides / Q000378:metabolism*
D003574:Cytochrome c Group; D045362:Cytochromes c2