Robert B. Gennis
Professor of Biochemistry, Biophysics, and Chemistry
Professor Robert B. Gennis received his undergraduate degree from the University of Chicago in 1966 and his Ph.D. from Columbia in 1971. Professor Gennis' research interests are in biochemistry and biophysical chemistry.
Research
Our laboratory is primarily engaged in studying membrane-bound metalloproteins that catalyze electron transfer reactions coupled to the generation of both a voltage and ion gradient across the membrane bilayer. Our goal is to determine the catalytic mechanisms and, in particular, the way by which these enzymes generate an electrochemical potential gradient across the membrane. Among the enzymes on which we are working are the respiratory oxidases from Escherichia coli and from 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. In addition we are studying the sodium pumping respiratory NADH:ubiquinone oxidoreductase from Vibrio cholerae. This organism uses a sodium electrochemical gradient generated by this enzyme to drive a number of energy requiring processes, such as flagellar rotation required for cell motility. Generally, our research centers around how redox chemistry is coupled to moving ions (protons or sodium) across a membrane.
Techniques we use include classical preparative biochemistry, immunology, genetics, molecular biology, and biophysical methods such as FTIR spectroscopy, electrochemistry and rapid kinetics. In the E. coli system, our efforts are directed at the two terminal oxidases: cytochrome bo3 and cytochrome bd. Both of these enzymes oxidize ubiquinol in the cytoplasmic membrane and reduce O2 to H2O. Both generate a transmembrane voltage during enzyme turnover. Whereas the active site of the heme-copper oxidases contains a heme and a copper atom, the corresponding site in the bd-type oxidases contains two hemes.
Cytochrome bo3 is a member of the heme-copper superfamily of respiratory oxidases and is closely related to the mitocondrial cytochrome oxidase. The aa3-type and the cbb3-type cytochrome c oxidases from Rhodobacter sphaeroides are also members of the heme-copper oxidase superfamily. These enzymes are proton pumps, coupling the oxygen chemistry catalyzed at the active site to the electrogenic movement of protons across the membrane. We are particularly interested in determining the way in which protons are transferred within these heme-copper oxidases and the mechanism by which the proton pump operates. The X-ray structure of cytochrome oxidase provides a framework for extensive structure/function studies. We have identified two functionally important proton-conducting pathways.
Publications
Accommodation of two diatomic molecules in cytochrome bo: insights into NO reductase activity in terminal oxidases. Hayashi T, Lin MT, Ganesan K, Chen Y, Fee JA, Gennis RB, Moënne-Loccoz P. Biochemistry. 2009 Feb 10;48(5):883-90.
Identification of the nitrogen donor hydrogen bonded with the semiquinone at the Q(H) site of the cytochrome bo3 from Escherichia coli. Lin MT, Samoilova RI, Gennis RB, Dikanov SA.J. Am Chem Soc. 2008 Nov 26;130(47):15768-9.
Cytochrome c oxidase: exciting progress and remaining mysteries. Brzezinski P, Gennis RB. J Bioenerg Biomembr. 2008 Oct;40(5):521-31. Epub 2008 Oct 31. Review.
Structural changes due to the deprotonation of the proton release group in the M-photointermediate of bacteriorhodopsin as revealed by time-resolved FTIR spectroscopy. Morgan JE, Vakkasoglu AS, Lugtenburg J, Gennis RB, Maeda A. Biochemistry. 2008 Nov 4;47(44):11598-605. Epub 2008 Oct 7.
The fully oxidized form of the cytochrome bd quinol oxidase from E. coli does not participate in the catalytic cycle: direct evidence from rapid kinetics studies. Yang K, Borisov VB, Konstantinov AA, Gennis RB. FEBS Lett. 2008 Oct 29;582(25-26):3705-9. Epub 2008 Sep 26.
Electron and proton transfer in the ba(3) oxidase from Thermus thermophilus. Smirnova IA, Zaslavsky D, Fee JA, Gennis RB, Brzezinski P. J Bioenerg Biomembr. 2008 Aug;40(4):281-7. Epub 2008 Aug 28.
A role for internal water molecules in proton affinity changes in the Schiff base and Asp85 for one-way proton transfer in bacteriorhodopsin. Morgan JE, Gennis RB, Maeda A. Photochem Photobiol. 2008 Jul-Aug;84(4):1038-45. Epub 2008 Jun 28.
Impaired proton pumping in cytochrome c oxidase upon structural alteration of the D pathway. Lepp H, Salomonsson L, Zhu JP, Gennis RB, Brzezinski P.Biochim Biophys Acta. 2008 Jul-Aug;1777(7-8):897-903. Epub 2008 Apr 16.
The lifetimes of Pharaonis phoborhodopsin signaling states depend on the rates of proton transfers--effects of hydrostatic pressure and stopped flow experiments. Kikukawa T, Saha CK, Balashov SP, Imasheva ES, Zaslavsky D, Gennis RB, Abe T, Kamo N. Photochem Photobiol. 2008 Jul-Aug;84(4):880-8. Epub 2008 Mar 12.
Activity of membrane proteins immobilized on surfaces as a function of packing density. Friedrich MG, Kirste VU, Zhu J, Gennis RB, Knoll W, Naumann RL. J Phys Chem B. 2008 Mar 13;112(10):3193-201. Epub 2008 Feb 19.
Chemical shift assignment of the transmembrane helices of DsbB, a 20-kDa integral membrane enzyme, by 3D magic-angle spinning NMR spectroscopy. Li Y, Berthold DA, Gennis RB, Rienstra CM. Protein Sci. 2008 Feb;17(2):199-204.
Strong excitonic interactions in the oxygen-reducing site of bd-type oxidase: the Fe-to-Fe distance between hemes d and b595 is 10 A. Arutyunyan AM, Borisov VB, Novoderezhkin VI, Ghaim J, Zhang J, Gennis RB, Konstantinov AA. Biochemistry. 2008 Feb 12;47(6):1752-9. Epub 2008 Jan 19.
Diversity of the heme-copper superfamily in archaea: insights from genomics and structural modeling. Hemp J, Gennis RB. Results Probl Cell Differ. 2008;45:1-31. Review.
A new ruthenium complex to study single-electron reduction of the pulsed O(H) state of detergent-solubilized cytochrome oxidase.Brand SE, Rajagukguk S, Ganesan K, Geren L, Fabian M, Han D, Gennis RB, Durham B, Millett F.Biochemistry. 2007 Dec 18;46(50):14610-8. Epub 2007 Nov 21.
Flash-photolysis of fully reduced and mixed-valence CO-bound Rhodobacter sphaeroides cytochrome c oxidase: heme spectral shifts.Szundi I, Ray J, Pawate A, Gennis RB, Einarsdóttir O.Biochemistry. 2007 Nov 6;46(44):12568-78. Epub 2007 Oct 12.
Comparative genomics and site-directed mutagenesis support the existence of only one input channel for protons in the C-family (cbb3 oxidase) of heme-copper oxygen reductases. Hemp J, Han H, Roh JH, Kaplan S, Martinez TJ, Gennis RB. Biochemistry. 2007 Sep 4;46(35):9963-72. Epub 2007 Aug 4.
Glutamate 107 in subunit I of the cytochrome bd quinol oxidase from Escherichia coli is protonated and near the heme d/heme b595 binuclear center. Yang K, Zhang J, Vakkasoglu AS, Hielscher R, Osborne JP, Hemp J, Miyoshi H, Hellwig P, Gennis RB. Biochemistry. 2007 Mar 20;46(11):3270-8. Epub 2007 Feb 17.
Water structural changes in the L and M photocycle intermediates of bacteriorhodopsin as revealed by time-resolved step-scan Fourier transform infrared (FTIR) spectroscopy. Morgan JE, Vakkasoglu AS, Gennis RB, Maeda A. Biochemistry. 2007 Mar 13;46(10):2787-96. Epub 2007 Feb 15.
An EPR spin label study of the quinol oxidase, E. coli cytochrome bo3: a search for redox induced conformational changes. White GF, Field S, Marritt S, Oganesyan VS, Gennis RB, Yap LL, Katsonouri A, Thomson AJ. Biochemistry. 2007 Mar 6;46(9):2355-63. Epub 2007 Feb 9.
Partial (13)C and (15)N chemical-shift assignments of the disulfide-bond-forming enzyme DsbB by 3D magic-angle spinning NMR spectroscopy. Li Y, Berthold DA, Frericks HL, Gennis RB, Rienstra CM. Chembiochem. 2007 Mar 5;8(4):434-42.
