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.