PATHWAY OF PROTON-TRANSFER IN BACTERIAL REACTION CENTERS - 2ND-SITE MUTATION ASN-M44-]ASP RESTORES ELECTRON AND PROTON-TRANSFER IN REACTION CENTERS FROM THE PHOTOSYNTHETICALLY DEFICIENT ASP-L213-]ASN MUTANT OF RHODOBACTER-SPHAEROIDES

Site-directed mutagenesis of the photosynthetic reaction center (RC) from Rhodobacter sphaeroides has shown Asp-213 of the L subunit (Asp-L213) to be important for photosynthetic viability. Replacement of Asp-L213 with Asn resulted in a photosynthetically deficient mutant, due to the 10(4)-fold slower rate for the proton-coupled electron transfer reaction Q(A)-Q(B)- + 2H+ --> Q(A)Q(B)H-2 (k(AB)(2)). The detrimental effect of Asn-L213 is surprising since RCs from Rhodopseudomonas viridis, Rhodospirillum rubrum, and Chloroflexus aurantiacus have Asn at the homologous position. However, RCs from these bacteria have an Asp located near Q(B) (the secondary quinone acceptor) at the position homologous to Asn-M44 in Rb. sphaeroides which might function in place of Asp-L213. To test this conjecture a ''viridis-like'' structure was introduced into Rb. sphaeroides by replacing Asp-L213 with Asn and Asn-M44 with Asp. The RCs from this double mutant displayed near-native rates for the electron transfer reaction k(AB)(2) and restored photosynthetic competence. The rates for the first electron transfer reaction Q(A)-Q(B) --> Q(A)Q(B)- (k(AB)(1)) and charge recombination D+Q(A)Q(B)- --> DQ(A)Q(B) (k(BD)) were also restored to near-native values. These results indicate that Asp at either the L213 or the M44 site near Q(B) can provide a pathway for rapid proton transfer and explain why Asp-L213 need not be conserved in different photosynthetic bacteria. To test further the effect of Asp at M44 on electron and proton transfer to Q(B) a mutant containing Asp at both L213 and M44 was constructed. The RCs from this mutant (Asn-M44 --> Asp) exhibited faster proton-coupled electron transfer to Q(B)-. The increased rate of proton-coupled electron transfer (k(AB)(2)) in the presence of negatively charged Asp residues near Q(B) suggests the role of an Asp near Q(B) as (i) a proton donor group in the proton transfer chain and/or (ii) a negatively charged residue stabilizing proton transfer to reduced Q(B).