Lawrence J. Prochaska, Ph.D.
Ph.D.: The Ohio State University, 1975 (E.L. Gross)
Postdoctorals: Purdue University (R.A. Dilley); University of Oregon (R.A. Capaldi)
Our laboratory studies the biochemistry and molecular biology of membrane-bound enzymes that are crucial in heart and bacterial energy conservation reactions. Our research focuses on structure/function relationships in heart mitochondrial and bacterial cytochrome c oxidases, using immunological, biochemical, biophysical, and recombinant DNA methods. Cytochrome c oxidase is the terminal member of the respiratory chain of the mitochondrion and some aerobic bacteria. The enzyme oxidizes cytochrome c and reduces molecular oxygen into water while conserving the energy of its redox reactions by the vectorial translocation of protons. The mitochondrial and bacterial forms of the enzyme have 13 and 4 subunits, respectively. Three dimensional crystal structures for both forms of the enzyme are known; however, the molecular mechanism of proton-pumping is currently unknown.
Our research uses the three dimensional structures to ascertain the path of protons through the enzyme complex. This is primarily done by site directed mutagenesis of conserved amino acid residues in the bacterial enzyme and determining the effects of the mutation on cytochrome c oxidase electron transfer and proton pumping activities of the enzyme reconstituted in phospholipid vesicles. The choice of amino acid residues which are mutated is based upon their conservation across species and through computational molecular modeling.
Our primary focus in the laboratory is on the role of subunit III of cytochrome c oxidase in the structure and function of the enzyme. Subunit III, which is a mitochondrial encoded subunit that is highly conserved between prokaryotes and eukaryotes, is a seven helical membrane spanning polypeptide and currently has an unknown function in the enzyme's activities. We have spent considerable effort assessing the role of this subunit in cytochrome c oxidase structure and function and our current hypothesis is that subunit III regulates the enzyme's activities by conformational change.
Other laboratory interests include: 1) Mechanism of integral membrane protein reconstitution into artificial membranes or liposomes; 2) Integral membrane protein-detergent interactions; 3) Functional oligomeric state of intergral membrane proteins in vitro and in vivo, including cytochrome c oxidase; 4) The effects of toxic chemicals on brain, heart, and muscle mitochondrial functioning; 5) The effects of ischemia on mitochondrial functioning in a heart failure model.
- Nguyen, X.-T., Pabarue, H. A., Geyer, R. R., Shroyer, L. A., Estey, L. A., Parilo, M., Wilson, K.S., and Prochaska, L. Purification of Phospholipid Vesicles Containing Control and Subunit III-Depleted Beef Heart Cytochrome c Oxidase, Protein Expression and Purification 26, 122-130 (2002).
- Lincoln, A.J., Donat, N., Palmer, G., and Prochaska, L. The Effects of Site-Specific Polyclonal Antibodies Against Conserved Hydrophilic Domains of Bovine Heart Cytochrome c Oxidase Subunit III on the Functioning of the Enzyme. Archives of Biochemistry and Biophysics, 416, 81-91 (2003).
- Riegler, D., Shroyer, L.A., Pokalsky, C., Zaslavsky, D., Gennis, R., and Prochaska, L.J. Characterization of Steady-state Activities of Cytochrome c Oxidase at Alkaline pH: Mimicking the Effect of K-channel Mutations in the Bovine Enzyme. Biochimica Biophysica acta, 1706, 126-133 (2005).
- Geyer, R. R, Patli, S. S., Alter, G. M., Hosler, J. P., and Prochaska. L. J., Cytochrome c Oxidase Subunit I FromRhodobacter sphaeroides Assumes an Alternative Conformation in the Absence of Subunit III. Submitted (2006).
- Cvetkov, T., Shroyer, L.A., and Prochaska, L. J. Purification of Phospholipid Vesicles Containing Cytochrome cOxidase from Rhodobacter sphaeroides. Submitted (2006).