Optimizing multi-step B-side charge separation in photosynthetic reaction centers from Rhodobacter capsulatus.

Using high-throughput methods for mutagenesis, protein isolation and charge-separation functionality, we have assayed 40 Rhodobacter capsulatus reaction center (RC) mutants for their P + Q B − yield (P is a dimer of bacteriochlorophylls and Q is a ubiquinone) as produced using the normally inactive B-side cofactors B B and H B (where B is a bacteriochlorophyll and H is a bacteriopheophytin). Two sets of mutants explore all possible residues at M131 (M polypeptide, native residue Val near H B ) in tandem with either a fixed His or a fixed Asn at L181 (L polypeptide, native residue Phe near B B ). A third set of mutants explores all possible residues at L181 with a fixed Glu at M131 that can form a hydrogen bond to H B . For each set of mutants, the results of a rapid millisecond screening assay that probes the yield of P + Q B − are compared among that set and to the other mutants reported here or previously. For a subset of eight mutants, the rate constants and yields of the individual B-side electron transfer processes are determined via transient absorption measurements spanning 100 fs to 50 μs. The resulting ranking of mutants for their yield of P + Q B − from ultrafast experiments is in good agreement with that obtained from the millisecond screening assay, further validating the efficient, high-throughput screen for B-side transmembrane charge separation. Results from mutants that individually show progress toward optimization of P + H B − → P + Q B − electron transfer or initial P* → P + H B − conversion highlight unmet challenges of optimizing both processes simultaneously. [ABSTRACT FROM AUTHOR]