Ohio Advanced EPR Laboratory at Miami University

A resource serving the pulsed and CW EPR needs of the greater Ohio area.

Michael W. Crowder Professor
Department of Chemistry and Biochemistry
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(513) 529-7274

Ph.D. University of Virginia 1993

The focus of my research group is the structural and mechanistic characterization of metalloenzymes. One system that we work on is the metallo-beta-lactamases, which are Zn(II)-metalloenzymes that hydrolyze beta-lactam containing antibiotics and render bacteria resistant to these drugs. We have developed a number of methods to replace the naturally-occurring Zn(II) with Co(II), which has a S = 3/2 spin state, and the Co(II)-substituted enzymes retain almost 100% of the activity and structure of the native enzymes. We have been using low temperature, cw-X-band EPR to probe the electronic structure of Co(II) when bound to the enzymes, and EPR spectroscopy, along with spectral simulations, can be used to monitor the number of ligands bound to Co(II) and to suggest whether there are solvent molecules bound to the metal center. These studies have also been used to probe whether metal ions, when part of a ligand-bridged dinuclear metal center, are spin-coupled. Recently in combination with rapid freeze techniques, we have been able to probe the metal center during catalysis, and these experiments have revealed the presence of an EPR-active reaction intermediate that is common among all metallo-beta-lactamases.

Cw-EPR has also been used to probe the dynamics of an alpha helix that sits above the active site in Co(II)-substituted metallo-beta-lactamase ImiS. ImiS was site-specifically spin labeled on the alpha helix, and EPR spectra demonstrate that the spin label couples to Co(II), resulting in a broadened EPR signal for the spin-label. Rapid freeze quench EPR samples of the enzyme mixed with substrate demonstrate that the spin label moves ca. 3 angstroms from Co(II) as substrate binds. These data suggest a mechanistic role for the alpha helix in catalysis.

We plan to probe the dynamics of the invariant loop that extends above the active site in metallo-beta-lactamases. This loop has been postulated to play a significant role in catalysis; specifically, this loop is thought to "clamp" down on the substrate during catalysis, thereby raising the ground state energy of the substrate and speeding up the hydrolysis reaction. We plan to probe the distance that the loop moves by using rapid-freeze quench methodology along with pulsed EPR studies. Each metallo-beta-lactamase will be site-specifically spin labeled, and the resulting enzymes will be reacted with substrate (and reaction quenched). DEER studies will be used to probe the spin-label/spin-label distance in the unreacted and reacted enzymes in an effort to measure how far the loops move during catalysis.

Miami University

Department of Chemistry and Biochemistry

Ohio Advanced EPR Laboratory
701 East High Street
101 Hughes Laboratories
Oxford, OH 45056

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