Mg coordination by amino acid side chains is not required for assembly and function of the special pair in bacterial photosynthetic reaction centers.

A conserved histidine serves as the axial ligand to the Mg of bacteriochlorophylls in the photosynthetic reaction center (RC) and many other photosynthetic systems. The histidine axial ligands to each and both bacteriochlorophylls of the special-pair primary electron donor of the Rhodobacter sphaeroides RC have been replaced with glycine to create a cavity. In each case, RCs assemble and a normal special-pair comprised of Mg-containing bacteriochlorophylls is formed, as judged by many different spectroscopic and functional probes (e.g., absorption and Stark spectra, *P decay kinetics, P+Q(A)- recombination rate, and the redox potential of P). In contrast with heme proteins, where this strategy has been exploited to introduce exogenous organic ligands that can greatly affect the functional properties of the protein [DePillis, G. D., Decatur, S. M., Barrick, D., & Boxer, S. G. (1994) J. Am. Chem. Soc. 116, 6981-6982], addition of exogenous imidazole, pyridine, and ethanethiol has no measurable effect on the functional properties of the special pair in these cavity mutants. FT-Raman spectroscopy is used to provide more detailed information on local interactions around the special pair. Data in the core-size marker mode and carbonyl stretching region suggest that an adventitious ligand replaces histidine as the axial ligand to bacteriochlorophylls in the cavity mutants. We speculate that this ligand is water. Furthermore, the position of the core-size marker mode changes when the cavity mutant RCs are incubated with exogenous ligands such as imidazole, pyridine, or ethanethiol, suggesting that the axial ligand to the special pair BChls can be exchanged in the cavity mutants. Interestingly the temperature dependence of P+Q(A)- recombination kinetics is very similar in the cavity mutants and WT, suggesting that the axial ligands to the special pair are not significant contributors to the solvent reorganization energy for this reaction. These results lead to the surprising conclusion that the nature of the axial ligand to the special pair has little influence on the properties of the macrocycle, and that axial coordination from the protein by histidine is not required for bacteriochlorophyll binding or for efficient electron transfer in the RC.