Quinone (Q_B) Reduction by B-Branch Electron Transfer in Mutant Bacterial Reaction Centers fromRhodobacter sphaeroides: Quantum Efficiency and X-ray Structure^,

Abstract

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 Q(A). Subsequent electron and proton transfer to a mobile quinone molecule Q(B) converts it to a quinol, Q(B)H(2). We report the construction and characterization of a series of mutants in Rhodobacter sphaeroides designed to reduce Q(B) via the B-branch. The quantum efficiency to Q(B) via the B-branch Phi(B) 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 Phi(B) obtained in the AW(M260) mutant, which lacks Q(A), to the 100% quantum efficiency for Phi(A) 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 A (R-factor = 19.6%). The Q(A) 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.

Trapped Conformational States of Semiquinone (D+•QB-•) Formed by B-Branch Electron Transfer at Low Temperature in Rhodobacter sphaeroides Reaction Centers

“…4 assuming that Φ A = 100% at 80K (61). This is within uncertainty the same as the "overall" value of 0.4% observed at room temperature (10). The light saturation curves for the LHM214/AWM260 and GDM203/AWM260 were the same (Fig.…”

“…capsulatus (48,62), but was not observed in the room temperature measurements on the Rb. sphaeroides mutant RCs used in this study (10).…”

“…The mutations are: Leu-M214 → His (48), Gly-M203 → Asp (49-51), Tyr-M210 → Phe and Phe-L181 → Tyr (48,52,53). The construction of this quintuple mutant was previously reported (10). In the quintuple mutant we could form Q B −• via B-branch electron transfer at room temperature (54) (54).…”

“…Since in the native RC Q B −• cannot be formed by electron transfer from Q A −• in samples frozen in the dark (5, 43), we developed a modified RC (Fig. 1) in which electron transfer to Q B occurred through the B-branch bacteriopheophytin (H B −• ) (10,(44)(45)(46)(47) which has a much greater driving force for electron transfer (12). This allows higher energy states of Q B −• to become populated (47).…”

The polyketide cyclase RemF from Streptomyces resistomycificus contains an unusual octahedral zinc binding site

“…None of these octahedral Zn binding sites contain a four His motif. This motif appears to be very unusual for octahedral metal binding sites in general as a search for similar arrangements of four histidine side chains as part of octahedral ligand geometries in metalloproteins resulted in only two more hits, the protein TM1459 of unknown function from T. maritima [26], and chain L of the reaction center from Rhodobacter sphaeroides [31]. However, none of these proteins contains zinc.…”