Consequences of saturation mutagenesis of the protein ligand to the B-side monomeric bacteriochlorophyll in reaction centers from Rhodobacter capsulatus

In bacterial reaction centers (RCs), photon-induced initial charge separation uses an A-side bacteriochlorophyll (BChl, B-A) and bacteriopheophytin (BPh, H-A), while the near-mirror image B-side B-B and H-B cofactors are inactive. Two new sets of Rhodobacter capsulatus RC mutants were designed, both bearing substitution of all amino acids for the native histidine M180 (M-polypeptide residue 180) ligand to the core Mg ion of B-B. Residues are identified that largely result in retention of a BChl in the B-B site (Asp, Ser, Pro, Gln, Asn, Gly, Cys, Lys, and Thr), ones that largely harbor the Mg-free BPh in the B-B site (Leu and Ile), and ones for which isolated RCs are comprised of a substantial mixture of these two RC types (Ala, Glu, Val, Met and, in one set, Arg). No protein was isolated when M180 is Trp, Tyr, Phe, or (in one set) Arg. These findings are corroborated by ground state spectra, pigment extractions, ultrafast transient absorption studies, and the yields of B-side transmembrane charge separation. The changes in coordination chemistries did not reveal an RC with sufficiently precise poising of the redox properties of the B-B-site cofactor to result in a high yield of B-side electron transfer to H-B. Insights are gleaned into the amino acid properties that support BChl in the B-B site and into the widely observed multi-exponential decay of the excited state of the primary electron donor. The results also have direct implications for tuning free energies of the charge-separated intermediates in RCs and mimetic systems.