Characterization of a symmetrized mutant RC with 42 residues from the QA site replacing residues in the Q(B) site.
The electron transfer reactions involving Q(A) and Q(B) were investigated in Rb. capsulatus RCs where the Q(B) site was mutated to contain 42 residues from the Q(A) site. The RCs have M220-M261 in the Q(A) site substituted for L193-L227 in the Q(B) site plus the M subunit second-site mutations, M144MI and M145AS, which had been found to restore the ability of the bacteria to grow photosynthetically. These mutants lack L210D, L212E, L213D, and L223S which have been previously shown to affect the electron transfer from Q(A) (-) to Q(B). Despite the large change in the Q(B) pocket, secondary quinone function still can be reconstituted. The UQ(4) dissociation constant for the Q(B) site in the mutant is only three times as large as in the wild type RCs. The rate of charge recombination (P(+)Q(A)Q(B) (-) --> PQ(A)Q(B)) (k (BP)) is reduced from 8.9 s(-1) in wild type RCs to 0.05s(-1) in the mutant, This indicates that Q(A)Q(B) (-) is stabilized relative to Q(A) (-)Q(B) by at least 60 meV more than in wild type protein. k (BP) is pH independent in the mutant RCs, while in wild type RCs k (BP) increases at alkaline pHs as reduction of Q(B) becomes energetically less favorable. Similar pH independent, slow k (BP) has been found in the L212EA/L213DA double mutant. The largest change found in the mutant is that the electron transfer from Q(A) (-) to Q(B) (k (AB) ((1)) approximately 14 s(-1)) is 3 orders of magnitude slower than in wild type RCs (10(4) s(-1)).
Li
J
J
Physics Department, Harvard University, 17 Oxford Street Jefferson Labs, Cambridge, MA, 02138, USA.
Coleman
W J
WJ
Youvan
D C
DC
Gunner
M R
MR