Quinone (QB) Reduction by B-Branch Electron Transfer in Mutant Bacterial Reaction Centers from Rhodobacter sphaeroides: Quantum Efficiency and X-ray Structure

• Published in: Biochemistry (Easton) 2005-05, Vol.44 (18), p.6920-6928
• Main Authors: Paddock, M. L, Chang, C, Xu, Q, Abresch, E. C, Axelrod, H. L, Feher, G, Okamura, M. Y
• Format: Article
• Language: English
• Subjects: Bacteriochlorophylls - chemistry; Bacteriochlorophylls - genetics; Bacteriochlorophylls - metabolism; Benzoquinones - antagonists & inhibitors; Benzoquinones - chemistry; Benzoquinones - metabolism; Binding Sites - genetics; Crystallization; Crystallography, X-Ray; Electron Transport - genetics; Kinetics; Lasers; Models, Chemical; Mutagenesis, Site-Directed; Oxidation-Reduction; Pheophytins - chemistry; Pheophytins - genetics; Pheophytins - metabolism; Photolysis; Photosynthetic Reaction Center Complex Proteins - genetics; Photosynthetic Reaction Center Complex Proteins - isolation & purification; Photosynthetic Reaction Center Complex Proteins - metabolism; Rhodobacter sphaeroides; Rhodobacter sphaeroides - genetics; Rhodobacter sphaeroides - growth & development; Rhodobacter sphaeroides - metabolism; Spectrophotometry
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi047559m

The photosynthetic reaction center (RC) from purple bacteria converts light into chemical energy. Although the RC shows two nearly structurally symmetric branches, A and B, light-induced electron transfer in the native RC occurs almost exclusively along the A-branch to a primary quinone electron acceptor QA. Subsequent electron and proton transfer to a mobile quinone molecule QB converts it to a quinol, QBH2. We report the construction and characterization of a series of mutants in Rhodobacter sphaeroides designed to reduce QB via the B-branch. The quantum efficiency to QB via the B-branch ΦB ranged from 0.4% in an RC containing the single mutation Ala-M260 → Trp to 5% in a quintuple mutant which includes in addition three mutations to inhibit transfer along the A-branch (Gly-M203 → Asp, Tyr-M210 → Phe, Leu-M214 → His) and one to promote transfer along the B-branch (Phe-L181 → Tyr). Comparing the value of 0.4% for ΦB obtained in the AW(M260) mutant, which lacks QA, to the 100% quantum efficiency for ΦA along the A-branch in the native RC, we obtain a ratio for A-branch to B-branch electron transfer of 250:1. We determined the structure of the most effective (quintuple) mutant RC at 2.25 Å (R-factor = 19.6%). The QA site did not contain a quinone but was occupied by the side chain of Trp-M260 and a Cl-. In this structure a nonfunctional quinone was found to occupy a new site near M258 and M268. The implications of this work to trap intermediate states are discussed.