James Allan Cowan

Research Description:

OUR RESEARCH interests cover a diverse range of topics in the fields of bioinorganic, bioorganic, and biophysical chemistry. A wide variety of techniques (including synthesis, spectroscopy, biochemistry, and molecular biology) are used to tackle important problems at the interface of chemistry and biology. These include, the understanding of enzymatic multielectron redox reactions, RNA/DNA structure and function, and mechanisms of gene regulation. Four problems that we are currently studying are described below.

(1) The elucidation of the mechanisms of enzymatic multielectron redox reactions is an intriguing problem, requiring an understanding of the interaction between several prosthetic groups (e.g., hemes, Fe-S clusters flavins). We are currently investigating the 6e- reduction of SO32- to S2- in sulfate-reducing bacteria. Spectroscopic and electrochemical information on the relative geometry and redox properties of siroheme and [Fe4S4] cluster centers, and the detection of transient reaction intermediates, will enable us to (a) establish the mechanistic details of the reaction pathway (b) determine, by site-directed mutagenesis, the role of the protein environment in binding and stabilizing substrates, and (c) understand the relationship between electron-transfer and atom-transfer chemistry. These enzymes also exhibit other interesting physical effects; for instance, magnetic exchange interactions between sirohemes and [Fe4S4] clusters that can be studied using NMR spectroscopy. Practical methods will include protein isolation and modification, EPR, NMR, electrochemistry, laser flash photolysis, Mossbauer spectroscopy, resonance Raman, and site-directed mutagenesis.

(2) High potential iron proteins possess a [Fe4S4] cluster with an unusually positive reduction potential. We are currently studying the structural features of the protein pocket that control the redox, electronic, and magnetic properties of the cluster; define chemical mechanisms of biological cluster formation; and the role of the cluster in defining tertiary structure and protein folding. Projects will include a number of techniques, including site-directed mutagenesis, electrochemistry, highfield 1H NMR, and EPR.

(3) E. coli ribonuclease H is structurally homologous to the RNase H domain of HIV reverse transcriptase. Both require magnesium ion as an essential cofactor. Although magnesium-dependent enzymes are ubiquitous in nucleic acid biochemistry, the role of these cofactors in orchestrating enzyme structure and activity is poorly understood. Our principal goals are the evaluation of the structural features of protein binding sites that regulate the kinetic and thermodynamic factors underlying magnesium binding and the implications for reaction mechanism, the structural details of the substrate recognition mechanism, and a mechanistic understanding of the reaction pathway. We possess an overproducing strain for the low molecular weight (Mr~17,600) ribonuclease H (courtesy of Dr. R.J. Crouch, NIH), and the crystallographic coordinates to 1.7 resolution (courtesy of Dr. W. Hendrickson, Columbia). Our experimental approach combines molecular biology, 25Mg, 13C, 15N, and 1H NMR, calorimetry, and reaction kinetics.

(4) In recent years, the general mechanisms of DNA transcription and translation have become fairly well defined. Attention is now turning to questions concerning the regulation and control of gene expression, and the general interaction of DNA and RNA with proteins. As chemists, we aim to approach these problems at the molecular level by studying the coordination environment, and the catalytic and structural role of metal ions that control the interactions of metalloregulatory enzymes, restriction enzymes, ligases, and kinases with RNA and DNA.