This blog describes the outcome of two scientific projects (ERMOE and MC2) related to the structure and function of molybdenzymes, and funded by the French National Agency of Research (ANR), over the period 2006-2015.
MC2 and ERMOE are led by the laboratory of “Bioénergétique et Ingénierie des Protéines” (CNRS, Marseille), in collaboration with the “laboratoire de Chimie Bactérienne” (CNRS, Marseille) and the lab of “Bioénergétique Cellulaire” (CEA, Cadarache).
Structure and function of bacterial molybdoenzymes of the DMSO reductase family
In living organisms, chemical reactions are catalyzed by very large biological molecules called « enzymes ». Their molecular weight is in the range 60 to 1000 kg/mol. They usually consist of various proteins assembled together, and they include some non-proteic parts (e.g. inorganic or organometallic metallic clusters) called cofactors. The business end of the enzyme, where the chemical reaction occurs, is a special cofactor called « active site », which is often buried deep in the protein and connected to the outside by structural elements that are used for transferring protons and electrons. We focussed on a family of bacterial enzymes characterized by having a particular active site, a molybdenum ion bound by a complex organic molecule.
Beyond the active site, the structures of the enzymes in this family are very diverse, and so is the number and nature of the other cofactors. What matters is that the mechanism of these enzymes is delocalized over a molecule whose size can be as large as 15nm. The enzymes in this family catalyze oxo transfer reactions, e.g. the reduction of nitrate or the oxidation of arsenite. These reactions are part of the main natural cycles and they are also very important in the metabolism of the bacteria that house the enzymes we studied. Not least, these catalysts can be used in biotech devices aimed at controlling the concentrations of contaminants in natural and humanly-impacted environments (arsenic). The mechanism of these enzymes is still obscure, and there is no understanding today of what determines substrate specificity in this family of enzymes.
A multidisciplinary approach based on the comparison of homologous enzymes, site directed mutagenesis and biophysical techniques.
The original aspect of our work comes from the focus on the comparison of similar enzymes, rather than the study of one in particular, and from the interdisciplinary nature of the collaboration. As model systems, we selected two nitrate reductases and two arsenite oxidases on the following criteria: (1) These four enzymes illustrate the diversity of reactivities, active site coordinations and quaternary structures in this family of enzymes. (2) The reactions they catalyze are related to environmental concerns. (3) Their 3D structures are known (two of them had actually been determined in teams that are directly involved in the project). (4) We are able to produce and purify the proteins in amounts compatible with material-demanding techniques (5) and to selectively modify them by using genetic engineering. We have studied these enzymes in an interdisciplinary approach combining biochemistry and site-directed mutagenesis with modern electrochemical techniques, advanced or time-resolved optical and magnetic spectroscopies (EPR, ENDOR, ESEEM, MCD…) and X-ray crystallography.
We focus on all aspects related to function: on the structural, electronic and redox properties of the active sites in these enzymes, on the dynamics of long-range, intramolecular electron and proton transfers, and on the intermolecular steps at the sites of interaction with quinones. We have for example been able to analyse in great details the quinone binding site in the membrane bound nitrate reductase, the evolutionary relations between arsenite oxidases and their close homologues, and we questioned the conclusions from previous investigations that interpreted spectroscopic signatures of the Mo in periplasmic nitrate reductase.
So far, we have reported these results in papers published in international journals (PNAS, J. Biol. Chem, Biochemistry, J. Am. Chem. Soc., Mol. Biol. Evol., J. Phys. Chem. B, Anal. Chem.) and book chapters. The number and the quality of these journals attest to the amount and interest of the results which we have acquired, published and presented in a number of national and international scientific meetings.