Bacterial reaction centers have a single nonheme iron that is located between two bound quinones, QA and QB, which are the primary and secondary electron acceptors during photosynthesis, respectively. In Rhodobacter sphaeroides, the iron is coordinated by four nitrogen atoms, contributed by histidines at L190, L230, M219, and M266, and two oxygen atoms, contributed by Glu at M234. The roles of these ligands in determining the metal-binding specificity and electron transfer properties of the quinones were investigated by mutagenesis. Each of the four His ligands was changed to Glu, Gln, and Cys, whereas Glu was changed to His, Gln, Cys, and Asp. All mutants supported photosynthetic growth except for those with substitutions of Glu or Cys at L190 or M219. The metal specificity of isolated mutant RCs was determined by measurements using atomic absorption and 35 GHz electron paramagnetic resonance spectroscopy. The M234 mutants had a lesser iron specificity than the wild type with a mole fraction of 0.7 to 0.8 iron but retained a total metal content of 1.0. All His mutants had an even lower iron content with mole fractions of 0.04 to 0.16. The His to Cys at M266 mutant had a significantly greater amount of bound zinc that was further enhanced when the strain was grown in zinc-supplemented media. The charge recombination rates from Q B-., which ranged from 0.5 to 1 s-1 in the mutants, were comparable to the 1 s-1 value for the wild type. Charge recombination from QA-. showed complex kinetics, with rates of 15 to 30 s-1 for the L190, L230, and M234 mutants and 200 s-1 for the M266 mutants compared with 8 s-1 for the wild type. The faster rates in the mutants most likely reflected a smaller free energy difference between QA-. and ΦA - , a nearby bacteriopheophytin, with the smaller energy difference facilitating indirect recombination. All of the mutants transferred electrons to the secondary quinone, with rates (1200 to 4700 s-1) comparable to that of the native (3700 s-1). The data demonstrate that neither the ligands nor the bound metal play a critical role in the electron transfer processes at the acceptor side.
ASJC Scopus subject areas
- Atomic and Molecular Physics, and Optics