P. Sebban et al., ELECTROSTATIC DOMINOES - LONG-DISTANCE PROPAGATION OF MUTATIONAL EFFECTS IN PHOTOSYNTHETIC REACTION CENTERS OF RHODOBACTER-CAPSULATUS, Biochemistry, 34(26), 1995, pp. 8390-8397
Two point mutants from the purple bacterium Rhodobacter capsulatus, bo
th modified in the M protein of the photosynthetic reaction center, ha
ve been studied by flash-induced absorbance spectroscopy. These strain
s carry either the M231Arg --> Leu or M43Asn --> Asp mutations, which
are located 9 and 15 Angstrom, respectively, from the terminal electro
n acceptor Q(B). In the wild-type Rb. sphaeroides structure, M231Arg i
s involved in a conserved salt bridge with H125Glu and H232Glu and M43
Asn is located among several polar residues that form or surround the
Q(B) binding site. These substitutions were originally uncovered in ph
enotypic revertants isolated from the photosynthetically incompetent L
212Glu-L213Asp --> Ala-Ala site-specific double mutant. As second-site
suppressor mutations, they have been shown to restore the proton tran
sfer function that is interrupted in the L212Ala-L213Ala double mutant
. The electrostatic effects that are induced in reaction centers by th
e M231Arg --> Leu and M43Asn --> Asp substitutions are roughly the sam
e in either the double-mutant or wild-type backgrounds. In a reaction
center that is otherwise wild type in sequence, they decrease the free
energy gap between the Q(A)(-) and Q(B)(-) states by 24 +/- 5 and 45
+/- 5 meV, respectively. The pH dependences of K-2, the Q(A)(-)Q(B) <-
> Q(A)Q(B)(-) equilibrium constant, are altered in reaction centers th
at carry either of these substitutions, revealing differences in the p
K(a)s of titratable groups compared to the wild type. These results co
nfirm that interactions among distant residues influence the electrost
atic potential in the immediate vicinity of Q(B) to ensure the efficie
nt conduction of protons through the protein matrix and their delivery
to the reduced quinone. It is possible that these influences are prop
agated over such large distances by mutation-induced realignments of s
alt bridges within a network of acidic and basic residues that is loca
ted in this region of the reaction center, which could serve as a rela
y mechanism to partially relocate the new negative charge much closer
to the quinone.