Abstract

The bacterial reaction center (RC) is a membrane bound protein that plays the central role in the conversion lectromagnetic radiation into chemical energy. The RC catalyzes the light driven electron transfer from cytochrome c2 to a quinone-Fe complex, that contains a primary quinone QA and a secondary quinone QB. The primary quinone QA, plays an important role in the initial electron transfer step. The secondary quinone QB plays a central role in the electron and proteon translocation process, acting as a two electron two proton acceptor species which is involved in the primary step of proton translocation. This proposal addresses the question of the molecular basis for the function of the quinones using the technique of site directed mutagenesis in the bacterium Rb. sphaeroides. By modifying amino acid residues near the Q binding sites and measuring the concomitant changes in activity we will identify the residues involved in the following functions: a) Quinone and herbicide binding. Residues near the quinone binding sites will be modified and the binding constants of quinone and herbicide will be determined. The results will give information about the nature of substrate and inhibitor binding and the mechanism of herbicide resistance. b) Electron transfer kinetics. Aromatic and charged amino acids will be modified and their effects on electron tyransfer determined. The results should give information about the mechanisms of electron transfer. c) Electrostatic stabilization of QQ -. Charged amino acid residues will be modified and the equilibrium between Q-A QB and QAQ-B will be determined. The results should give information about the electrostatic interactions in a membrane protein. d) Proton transport to QB. Protonable residues which can potentialy form a proton transfer chain from the RC surface to the QB site will be modified and their effect on the rate of proton transfer will be detemined. The results should test the mechanism of proton transport in biological membranes.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM041637-03
Application #
3299927
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1989-04-01
Project End
1994-03-31
Budget Start
1991-04-01
Budget End
1992-03-31
Support Year
3
Fiscal Year
1991
Total Cost
Indirect Cost

  Institution

Name
University of California San Diego
Department
Type
Schools of Arts and Sciences
DUNS #
077758407
City
La Jolla
State
CA
Country
United States
Zip Code
92093

  Publications

Nechushtai, Rachel; Conlan, Andrea R; Harir, Yael et al. (2012) Characterization of Arabidopsis NEET reveals an ancient role for NEET proteins in iron metabolism. Plant Cell 24:2139-54
Flores, Marco; Okamura, Melvin Y; Niklas, Jens et al. (2012) Pulse Q-band EPR and ENDOR spectroscopies of the photochemically generated monoprotonated benzosemiquinone radical in frozen alcoholic solution. J Phys Chem B 116:8890-900
Zuris, John A; Ali, Syed S; Yeh, Howard et al. (2012) NADPH inhibits [2Fe-2S] cluster protein transfer from diabetes drug target MitoNEET to an apo-acceptor protein. J Biol Chem 287:11649-55
Zuris, John A; Harir, Yael; Conlan, Andrea R et al. (2011) Facile transfer of [2Fe-2S] clusters from the diabetes drug target mitoNEET to an apo-acceptor protein. Proc Natl Acad Sci U S A 108:13047-52
Conlan, Andrea R; Paddock, Mark L; Homer, Christina et al. (2011) Mutation of the His ligand in mitoNEET stabilizes the 2Fe-2S cluster despite conformational heterogeneity in the ligand environment. Acta Crystallogr D Biol Crystallogr 67:516-23
Zuris, John A; Halim, Danny A; Conlan, Andrea R et al. (2010) Engineering the redox potential over a wide range within a new class of FeS proteins. J Am Chem Soc 132:13120-2
Flores, Marco; Savitsky, Anton; Paddock, Mark L et al. (2010) Electron-nuclear and electron-electron double resonance spectroscopies show that the primary quinone acceptor QA in reaction centers from photosynthetic bacteria Rhodobacter sphaeroides remains in the same orientation upon light-induced reduction. J Phys Chem B 114:16894-901
Dicus, Michelle M; Conlan, Andrea; Nechushtai, Rachel et al. (2010) Binding of histidine in the (Cys)3(His)1-coordinated [2Fe-2S] cluster of human mitoNEET. J Am Chem Soc 132:2037-49
Paddock, M L; Flores, M; Isaacson, R et al. (2010) EPR and ENDOR Investigation of Rhodosemiquinone in Bacterial Reaction Centers Formed by B-Branch Electron Transfer. Appl Magn Reson 37:39
Iwata, Tatsuya; Paddock, Mark L; Okamura, Melvin Y et al. (2009) Identification of FTIR bands due to internal water molecules around the quinone binding sites in the reaction center from Rhodobacter sphaeroides. Biochemistry 48:1220-9

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