MC2 (2011-2015)

“MC2” stands for “Molybdenum Cofactor Chemistry”. It is a long term project aimed at understanding the mechanism of molybdoenzymes of the DMSO reductase family, funded by the French Agence Nationale de la Recherche (ANR), which follows up a former project caller ERMOE described here.

The 20 permanent researchers involved in the present project belong to three CNRS or CEA laboratories and represent the main French research groups working on the mechanism of molybdoenzymes. We have collaborated for the last four years within the framework of a previous ANR project (PCV 2006-2009). Our goal was to use a multidisciplinary approach to study all the molecular aspects of the reactivity of three complex (i.e. multicentered) enzymes of the so-called “DMSO reductase family” of molybdoenzymes. dmsoreductasefamily2

These enzymes share a similar mononuclear molybdenum cofactor (Moco), and are frequently involved in oxo transfer reactions: they may catalyse the reduction of nitrate, DMSO, chlorate, arsenate or selenate, or the oxidation of arsenite or nitrite. The toxicity of some of these substrates (chlorate, selenate and arsenite) in both natural, and humanly-impacted environments is an important issue of current public health. In the PCV project, we specifically studied the membrane bound, trimeric nitrate reductase from E. coli, the periplasmic, dimeric nitrate reductase from R. sphaeroides and the dimeric arsenite oxidase from Ralstonia sp. 22, using various biological and biophysical methods. We succeeded in tackling various aspects of the mechanism of the three enzymes of interest and we provided an integrated multi-disciplinary training to three young researchers. We challenged earlier conclusions according to which certain spectroscopically characterized species are catalytic intermediates, and we showed that the inability to generate species in which a substrate moiety is bound to the molybdenum ion is a major obstacle in mechanistic studies.

This led us to identify new questions and issues which will be addressed in the MC2 project: our goal is now to focus on the structure and function of the molybdenum cofactor, to find out what determines the substrate specificity of these homologous enzymes whose active sites are structurally very similar.

To achieve this goal, we have increased to 6 the number of enzymes we want to study, in order to cover a variety of active sites structures and functions and to make it possible to probe specific aspects of the reactivity. We have already acquired several new pieces of equipments (a freeze quench device for trapping intermediates in the milli-second time scales, several spectrometers (MCD and pulsed EPR) for characterizing them, a robot for optimizing crystallisation conditions and preparing oriented samples etc.). We have designed a work program that benefits from the diverse expertise of the participants in biochemistry and molecular biology, X-ray crystallography, kinetics including dynamic electrochemistry, advanced magnetic and optical spectroscopy, time-resolved spectroscopy, theoretical chemistry. We will use these methods for trapping and analysing the structure and kinetic properties of the catalytic intermediates of the six homologous enzymes of interest, and we will test the effect of modifying all the potential structural determinants of substrate specificity and directionality. Several academic recruits have already joined the new MC2 consortium, which will again bring together most of the French researchers working on the mechanism of molybdoenzymes, at the interface between biology, physics and chemistry.

Our investigations will bring an entire set of new data that will increase greatly our understanding of the reactivity of MOEs and of the determinants of function. Ultimately, we should be able to use site-directed mutagenesis to engineer nitrite or arsenite oxidase functions into a nitrate reductase “template”. The fact that all redox enzymes that exhibit a specific activity towards toxic oxides are close cousins of bacterial nitrate reductase suggests that the latter will be an ideal starting point for the rational design of biological remediation activity.

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