Potentiation of proton transfer function by electrostatic interactions in photosynthetic reaction centers from Rhodobacter sphaeroides: First results from site-directed mutation of the H subunit

The x-ray crystallographic structure of the photosynthetic reaction center (RC) has proven critical in understanding biological electron transfer processes, By contrast, understanding of intraprotein proton transfer is easily lost in the immense richness of the details, In the RC of Rhodobacter (Rb,) sphaeroides, the secondary quinone (Q(B)) is surrounded by amino acid residues of the L subunit and some buried water molecules, with M- and H-subunit residues also close by, The effects of site-directed mutagenesis upon RC turnover and quinone function have implicated several L-subunit residues in proton delivery to Q(B), although some species differences exist, In wild-type Rb. sphaeroides, Glu(L212) and Asp(L213) represent an inner shell of residues of particular importance in proton transfer to Q(B). Asp(L213) is crucial for delivery of the first proton, coupled to transfer of the second electron, while Glu(L212), possibly together with Asp(L213), is necessary for delivery of the second proton, after the second electron transfer, We report here the first study, by site-directed mutagenesis, of the role of the H subunit in Q(B) function, Glu(H173), one of a cluster of strongly interacting residues near Q(B), including Asp(L213), was altered to Gln. In isolated mutant RCs, the kinetics of the first electron transfer, leading to formation of the semiquinone, Q(B)(-), and the proton-linked second electron transfer, leading to the formation of fully reduced quinol, were both greatly retarded, as observed previously in the Asp(L213) --> Asn mutant, However, the first electron transfer equilibrium, Q(A)(-)Q(B) reversible arrow Q(A)Q(B)(-), was decreased, which is opposite to the effect of the Asp(L213) --> Asn mutation, These major disruptions of events coupled to proton delivery to QB were largely reversed by the addition of azide (N-3(-)). The results support a major role for electrostatic interactions between charged groups in determining the protonation state of certain entities, thereby controlling the rate of the second electron transfer, II is suggested that the essential electrostatic effect may be to ''potentiate'' proton transfer activity by raising the pK of functional entities that actually transfer protons in a coupled fashion with the second electron transfer, Candidates include buried water (H3O+) and Ser(L223) (serine-OH2+), which is very close to the O5 carbonyl of the quinone.