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Lawrence J. Prochaska, Ph.D.
Professor
Lab: 004 Diggs Lab
Office: 060 Diggs Lab
(937) 775-2551
Email Dr. Prochaska
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Education:
Ph.D.: The Ohio State University, 1975 (E.L. Gross)
Postdoctorals: Purdue University (R.A. Dilley); University of Oregon
(R.A. Capaldi)
Research Interests:
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.
Recent Publications:
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 From Rhodobacter
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 c Oxidase from Rhodobacter
sphaeroides. Submitted (2006). |
