The coupling of protein-mediated electron and proton transfer across a membrane of a cell or organelle is essential for life as it is the means by which biological systems establish an electrochemical gradient that can be used for the generation and storage of energy in the form of ATP. The proposed research will discover fundamental structural factors that underlie the bioenergetics and efficiency of electron-coupled proton transfer in transmembrane proteins. The power of our approach comes from the ability to correlate high-resolution structures of the bacterial photosynthetic reaction center (RC) with a wealth of spectroscopic data characterizing function of native and mutant RC complexes. This strategy is complemented by the ability to manipulate the physiology of Rhodobacter to apply selective pressure for 'repair' of engineered RCs whose function is impaired. Using x-ray diffraction of single crystals, we will determine the structures of RCs from R. sphaeroides carrying mutations that control proton transfer to the secondary quinone QB. The main emphasis of this project will be structural characterization of a panel of RCs derived from phenotypic revertants of engineered strains that are photosynthetically incompetent. RCs carrying the engineered mutations are incapable of transferring the first and/or second proton to reduced QB. Biophysical studies have determined that second-site compensatory mutations - some of which are quite distant from QB or the site of the original engineered substitutions - in the phenotypic revertants restore function to the RCs by activating alternative proton delivery pathways. In preliminary studies, we have determined the structure of one functionally impaired mutant RC (Pokkuluri et al., Biochemistry 41: 5998-6007, 2002). We are now in the process of determining the structures of two RCs derived from phenotypic revertants of it, from crystals that diffract to 2.7-2.8 A. The distant compensatory mutations influence the positions and properties of residues near Q8 through correlated motions of neighboring amino acid side chains. We have observed that these changes in main chain positions and side chain orientations can extend up to 40 angstrom from the site of the compensatory mutation. Data collected from these crystals of mutant and revertant RCs resulted in excellent electron density maps that clearly showed several unexpected effects of the mutations. The current panel of about 10 phenotypic revertants that we propose to study contains physiochemically diverse compensatory amino acid changes at sites both close to and distant from the apparent proton transfer pathway used within the native RC. By correlating the crystallographic data with the spectroscopic data, we will recognize the structural elements that constitute a functional, efficient proton transfer pathway; therefore, we will be able to discern similar elements in other proteins for which transmembrane proton translocation is a common feature.
| Pokkuluri, P Raj; Laible, Philip D; Crawford, Adam E et al. (2004) Temperature and cryoprotectant influence secondary quinone binding position in bacterial reaction centers. FEBS Lett 570:171-4 |
