High Yield of M-Side Electron Transfer in Mutants of Rhodobacter capsulatus Reaction Centers Lacking the L-Side Bacteriopheophytin

Abstract

We present studies on a series of photosynthetic reaction center (RC) mutants created in the background of the Rhodobacter capsulatus D(LL) mutant, in which the D helix of the M subunit has been substituted with that from the L subunit. Previous work on the D(LL) mutant in chromatophore preparations showed that RCs assembled without the bacteriopheophytin H(L) electron acceptor and performed no charge separation following light absorption. We have successfully isolated poly-His-tagged D(LL) RCs by using the detergent Deriphat 160-C and shown that the RCs are devoid of H(L). The excited state of the primary electron donor, P*, is found to have a lifetime of 180 +/- 20 ps and to decay exclusively (>95%) via internal conversion to the ground state, with no evidence for formation of any charge-separated intermediates. By additional mutation in the D(LL) background of two residues that affect the P/P+ oxidation potential and one that facilitates M-side electron transfer, we achieve an unprecedented 70% yield of P+ H(M)-, more than doubling the highest yield of this state achieved previously. This result underscores the importance of the relative free energies of P* and the charge-separated states in governing the rates and yields of electron transfer in bacterial RCs and provides a basis for systematically investigating M-side electron transfer without any competition from the native L-side pathway.

Temperature Dependence of Electron Transfer to the M-Side Bacteriopheophytin in Rhodobacter capsulatus Reaction Centers

“…Wild-type, D LL , and D LL -FY L F M RCs were expressed, isolated, and purified using the detergent Deriphat 160-C as described previously. 89 For measurements of D LL RCs in LDAO, the detergent was exchanged to LDAO after initial Deriphat 160-C purification. Due to extremely low yields, D LL -FY L F M was not subjected to buffer exchange and so was not examined in LDAO.…”

“…We previously showed that for D LL -FY L F M in Deriphat at 295 K, P* decay is well described by a single exponential (∼55 ps time constant) and gives a 70% yield of P + H M -and a 30% yield of deactivation to the ground state. 89 These values give a rate constant for electron transfer from P* to P + H M -of 0.7/(55 ps) ) (78 ps)…”

High Throughput Engineering to Revitalize a Vestigial Electron Transfer Pathway in Bacterial Photosynthetic Reaction Centers

“…Although significant yields of P ϩ H B Ϫ formation have been reported in some R. capsulatus mutants (17,31,32,35,38), the largest rate constant for P* 3 P ϩ H B Ϫ is ϳ(40 ps)…”

“…Further characterization of primary electron transfer events in select mutant RCs utilized a transient absorption apparatus having 130-fs excitation and white light probe flashes operating at 10 Hz (described previously in Refs. 31,38). RCs were held at ϳ10°C in an ice-cooled reservoir and flowed through a 2-mm pathlength cell.…”

“…The highest yields of ET from the excited dimer (P*) to H B (thus producing P ϩ H B Ϫ ) have been achieved in mutant Rhodobacter (R.) capsulatus RCs (17,31,32,35,38). In mutant RCs lacking Q A , reduction of Q B via sole activity of the B branch cofactors has been observed unambiguously (16, 24, 35, 37, 39 -41).…”