Robert B. Gennis

Department of Biochemistry and Chemistry
Ph.D. (1971) Columbia University

(217) 333-9075       r-gennis@uiuc.edu

Research Topics

• Structure and Mechanism of Proton-Pumping Respiratory Oxidases
• Biochemical & Biophysical Studies on the E. coli, V. cholera and R. sphaeroides Aerobic Respiratory Chains
• Application of Site-Directed Mutagenesis to Enzyme Structure/Function
• Membrane Protein Structure and Enzymology
• FTIR Spectroscopy

Research Interests

Our laboratory is primarily engaged in studying membrane-bound metalloproteins that catalyze electron transfer reactions coupled to the generation of both a voltage and a proton gradient across the membrane bilayer. Our goal is to determine the structure and catalytic mechanism at each of the redox active sites and, also, to determine the way by which these enzymes generate a proton electrochemical gradient across the membrane. The enzymes on which we are working are components of the aerobic respiratory chain of Escherichia coli, Vibro cholera or of Rhodobacter sphaeroides. The proton electrochemical gradients generated by these enzymes are used by the bacteria to provide the energy for active uptake of solutes and for the synthesis of ATP. The V. cholera respiratory chain is of interest because it generates a sodium gradient across the membrane.

Our approach is to apply a variety of techniques to this problem. These include classical preparative biochemistry, immunology, genetics, molecular biology, and biophysical methods. In the E. coli system, our efforts are primarily directed at characterizing the two cytochrome complexes that function as terminal oxidases. Both oxidases have been purified to homogeneity. The cytochrome bd complex contains three heme components and has two polypeptides by SDS-PAGE analysis. The cytochrome bo$$₃ complex has two heme components, copper, and four polypeptides. Both enzymes have been reconstituted in artificial proteoliposomes and shown to catalyze electrogenic reactions. That is, electron flow through the complexes results in generating a voltage difference across the bilayer. Both enzymes act as coupling sites in E. coli in that the reactions result in net proton translocation across the bilayer in vivo and in the reconstituted systems.

The genes encoding the cytochrome bd and bo$$₃ complexes have been cloned and the sequences of the genes encoding both have been determined. The sequences of the subunits of the cytochrome bo$$₃ complex have revealed a striking similarity with the mitochondrial cytochrome c oxidase, thus defining a superfamily of related respiratory oxidases. The aa₃ --type cytochrome c oxidase Rhodobacter sphaeroides, on which we also work, is another member of the heme-copper oxidase superfamily. Site-directed mutations and FTIR difference spectroscopy are being used to define residues involved in proton movements across the membrane. Our primary goal is to determine how this enzyme functions as a proton pump. This work is greatly assisted by the recent determination of the high resolution structures of two heme-copper oxidases.

In V. cholera we are studying a primary sodium pump which is an NADH: ubiquinone oxidoreductase.

Key Words: Recombinant DNA, Microbial Physiology, Site-Directed Mutagenesis, Membrane Structure and Function, Protein Purification and Characterization, Photosynthesis and Biophysical Techniques, Vibrio cholera

Current Research Funding: DOE, NIH, Human Frontier Science Program

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