In Situ, Protein-Mediated Generation of a Photochemically Active Chlorophyll Analogue in a Mutant Bacterial Photosynthetic Reaction Center.

All possible natural amino acids have been substituted for the native LeuL185 positioned near the B-side bacteriopheophytin (H _B ) in the bacterial reaction center (RC) from Rhodobacter sphaeroides . Additional mutations that enhance electron transfer to the normally inactive B-side cofactors are present. Approximately half of the isolated RCs with Glu at L185 contain a magnesium chlorin (C _B ) in place of H _B . The chlorin is not the common BChl a oxidation product 3-desvinyl-3-acetyl chlorophyll a with a C-C bond in ring D and a C═C bond in ring B but has properties consistent with reversal of these bond orders, giving 17,18-didehydro BChl a . In such RCs, charge-separated state P ⁺ C _B ^- forms in ∼5% yield. The other half of the GluL185-containing RCs have a bacteriochlorophyll a (BChl a ) denoted β _B in place of H _B . Residues His, Asp, Asn, and Gln at L185 yield RCs with ≥85% β _B in the H _B site, while most other amino acids result in RCs that retain H _B (≥95%). To the best of our knowledge, neither bacterial RCs that harbor five BChl a molecules and one chlorophyll analogue nor those with six BChl a molecules have been reported previously. The finding that altering the local environment within a cofactor binding site of a transmembrane complex leads to in situ generation of a photoactive chlorin with an unusual ring oxidation pattern suggests new strategies for amino acid control over pigment type at specific sites in photosynthetic proteins.