title: Pathway of proton transfer in bacterial reaction centers: replacement of serine-L223 by alanine inhibits electron and proton transfers associated with reduction of quinone to dihydroquinone.
abstract: The pathway of proton transfer in the reaction center (RC) from Rhodobacter sphaeroides was investigated by site-directed mutagenesis. Ser-L223, a putative proton donor that forms a hydrogen bond with the secondary quinone acceptor QB, was replaced with Ala and Thr. RCs with Ala-L223 displayed reduced electron transfer and proton uptake rates in the reaction QA-QB- + 2H+----QAQBH2. The rate constant for this reaction, k(2)AB, was found to be reduced approximately 350-fold to 4.0 +/- 0.2 s-1. Proton uptake measurements using a pH indicator dye showed a rapid uptake of 1 H+ per RC followed by a slower uptake of 1 H+ per RC at a rate of 4.1 +/- 0.1 s-1; native RCs showed a rapid uptake of 2H+ per RC. Evidence is provided that these changes were not due to gross structural changes in the binding site of QB. RCs with Thr-L223 showed little reduction in the rates of electron and proton transfer. These results indicate that proton transfer from the hydroxyl group of Ser-L223 or Thr-L223 is required for fast electron and proton transfer associated with the formation of the dihydroquinone QH2. In contrast, previous work showed that replacing Glu-L212, another putative proton donor to QB, with Gln slowed proton uptake from solution without significantly altering electron transfer. We propose a model that involves two distinct proton transfer steps. The first step occurs prior to transfer of the second electron to QB and involves proton transfer from Ser-L223. The second step occurs after this electron transfer through a pathway involving Glu-L212.
authors.affiliation: Department of Physics, University of California, San Diego, La Jolla 92093.
authors.forename: M L
authors.initials: ML
authors.lastname: Paddock
authors.forename: P H
authors.initials: PH
authors.lastname: McPherson
authors.forename: G
authors.initials: G
authors.lastname: Feher
authors.forename: M Y
authors.initials: MY
authors.lastname: Okamura
mesh_terms: D000409:Alanine; D016227:Benzoquinones; D002734:Chlorophyll / Q000235:genetics / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D006873:Hydroquinones / Q000378:metabolism*; D007700:Kinetics; D045342:Light-Harvesting Protein Complexes; D009154:Mutation; D010084:Oxidation-Reduction; D010788:Photosynthesis; D045322:Photosynthetic Reaction Center Complex Proteins; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011522:Protons; D011809:Quinones / Q000378:metabolism*; D012242:Rhodobacter sphaeroides / Q000235:genetics / Q000378:metabolism*; D012694:Serine
chemical_list: D016227:Benzoquinones; D006873:Hydroquinones; D045342:Light-Harvesting Protein Complexes; D045322:Photosynthetic Reaction Center Complex Proteins; D010940:Plant Proteins; D011522:Protons; D011809:Quinones; D002734:Chlorophyll; C004532:quinone; D012694:Serine; D000409:Alanine