Research Associate Professor, Department of Chemistry and Biochemistry
The tools of molecular genetics are used in our research to investigate the roles of proteins involved in photosynthesis. One particular focus of the research is the reaction center, which is the site of the primary reactions in photosynthesis. We can study in detail the relationship between the three-dimensional structure of the protein and its function in the reaction center from purple bacteria by using site-directed mutagenesis to design fairly precise and speciŪc changes in the protein. For example, we have engineered alterations in interactions such as metal ligands and hydrogen bonds between amino acid residues and the cofactors of the reaction center, which include bacteriochlorophylls, quinones, and an iron atom. Since many of the cofactors have distinct spectroscopic signatures, a variety of biochemical and biophysical techniques can be used to assay the effects of the mutations. The reaction center is involved in light-initiated electron transfer reactions, so it is of special interest to determine changes in the midpoint potentials of the cofactors, as this is an important parameter for determining the rate of electron transfer. An example of this type of study concerns the addition of hydrogen bonds from amino acid residues to the conjugated carbonyls of bacteriochlorophylls in the reaction center, where we have shown that the addition of a hydrogen bond can result in a substantial increase in the oxidation potential of the bacteriochlorophyll, with correlated changes in electron transfer rates.
Molecular genetics is also being used to explore the functions of less well-characterized proteins. Photosynthetic bacteria are rich in pigmented proteins that are amenable to such manipulation, giving rise to such projects as the isolation and sequence determination of the genes for reaction centers and cytochromes from novel bacteria and genetic analysis of the synthesis and assembly of pigment-protein complexes. Once a protein is targeted, we can isolate the gene, determine its sequence, construct strains in which the chromosomal gene is deleted, express the gene from a plasmid, and then initiate mutagenesis studies. This is done in conjunction with isolation of the protein, biochemical characterization of the properties of the protein including the cofactor composition and molecular weight, spectroscopic studies aimed at determining the rates of electron transfer or interactions with other proteins, and in some cases structural determination by X-ray diffraction of crystals. This array of techniques allows a diverse approach to the study of how proteins transduce energy.
Allen, J.P. and Williams, J.C. (1995) Relationship between the oxidation potential of the bacteriochlorophyll dimer and electron transfer in photosynthetic reaction centers. J. Bioenerg. Biomemb. 27, 275283.
Wang, S., Li, X., Williams, J.C., Allen, J.P. and Mathis, P. (1994) Interaction between cytochrome c2 and reaction centers from purple bacteria. Biochemistry 33: 83068312.
Lin, X., Murchison, H.A., Nagarajan, V., Parson, W.W., Allen, J.P. and Williams, J.C. (1994) SpeciŪc alteration of the oxidation potential of the electron donor in reaction centers form Rhodobacter sphaeroides. Proc. Natl. Acad. Sci. USA 91: 1026510269.
Murchison, H.A., Alden, R.G., Allen, J.P., Peloquin, J.M., Taguchi, A.K.W., Woodbury, N.W. and Williams, J.C. (1993) Mutations designed to modify the environment of the primary electron donor of the reaction center form Rhodobacter sphaeroides: Phenylalanine to leucine at L167 and histidine to phenylalanine at L168. Biochemistry 32: 34983505.
Williams, J.C., Alden, R.G., Murchison, H.A., Peloquin, J.M., Woodbury, N.W. and Allen, J.P. (1992) Effects of mutations near the bacteriochlorophylls in reaction centers from Rhodobacter sphaeroides. Biochemistry 31: 1102911037.
Tel: 1-(480) 965-0455
Fax: 1-(480) 965-2747
Office Room Number: Physical Sciences PS D-217
Lab Room Number: Physical Sciences PS C-134/PS C-44
Arizona State University
Room PSD 209
Tempe, AZ 85287-1604
01 February 2006
phone: (480) 965-1963
fax: (480) 965-2747