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.
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.
Paddock
M L
ML
Department of Physics, University of California, San Diego, La Jolla 92093.
McPherson
P H
PH
Feher
G
G
Okamura
M Y
MY
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
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