Reaction of cytochromes c and c2 with the Rhodobacter sphaeroides reaction center involves the heme crevice domain.
In order to define the interaction domain on Rhodobacter sphaeroides cytochrome c2 for the photosynthetic reaction center, positively charged lysine amino groups on cytochrome c2 were modified to form negatively charged (carboxydinitrophenyl)- (CDNP-) lysines. The reaction mixture was separated into several different fractions by ion-exchange chromatography on (carboxymethyl)cellulose. Tryptic digests of these fractions were analyzed by reverse-phase peptide mapping to determine the lysines that had been modified. Fraction A was found to consist of a mixture of singly labeled derivatives modified at lysine-35, -88, -95, -97, and -105 and several other unidentified lysines comprising 32% of the total. Although it was not possible to resolve these derivatives, all of the identified lysines are located on the front surface of cytochrome c2 near the heme crevice. The second-order rate constant for the reaction of native cytochrome c2 with reaction centers was 2.0 X 10(8) M-1 s-1, while that for fraction A was 20-fold less, 1.0 X 10(7) M-1 s-1. This suggests that lysines surrounding the heme crevice of cytochrome c2 are involved in electrostatic interactions with carboxylate groups at the binding site of the reaction center. The reaction rates of horse heart cytochrome c derivatives modified at single lysine amino groups with trifluoroacetyl or trifluoromethylphenylcarbamoyl were also measured. Modification of lysine-8, -13, -27, -72, -79, and -87 surrounding the heme crevice significantly lowered the rate of reaction, while modification of lysines in other regions had no effect. This indicates that the reaction of horse heart cytochrome c with the reaction center also involves the heme crevice domain.
Hall
J
J
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville 72701.
Zha
X H
XH
Durham
B
B
O'Brien
P
P
Vieira
B
B
Davis
D
D
Okamura
M
M
Millett
F
F
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D003574:Cytochrome c Group / Q000378:metabolism*; D045362:Cytochromes c2; D006418:Heme / Q000378:metabolism*; D006736:Horses; D007700:Kinetics; D007834:Lasers; D008239:Lysine; D045322:Photosynthetic Reaction Center Complex Proteins; D011485:Protein Binding; D011487:Protein Conformation; D012242:Rhodobacter sphaeroides / Q000378:metabolism*
D001426:Bacterial Proteins; D003574:Cytochrome c Group; D045322:Photosynthetic Reaction Center Complex Proteins; D006418:Heme; D045362:Cytochromes c2; D008239:Lysine