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In situ conversion of recalcitrant petroleum sources, such as heavy oil, oil sands, and asphalts, to methane by microorganisms would have significant environmental and economic advantages over current mining and extraction methods. For such an approach to become practical, an understanding of the natural biogeochemical cycles in such environments is required, including the roles and characteristics of the indigenous microorganisms.

Toward that end, we are investigating mechanisms of metal uptake and regulation in Methanocorpusculum labreanum Z, a methanogenic archaeon from the Tar Pits at Rancho La Brea, Los Angeles.  Our focus has been a putative MarR-family protein, the gene for which we have cloned into E. coli for overexpression.

In this second year of the funded project, we have continued our purification and characterization of M. labreanum MarR.  While the protein expresses well, initial efforts to purify MarR resulted in irreversible oxidation and the formation of insoluble aggregates.  Technical modifications such that the protein preparation and subsequent purification now are performed in an anaerobic environment have been very successful.  The protein purified in this way has a UV-vis absorption spectrum consistent with the binding of an iron-sulfur cluster, and we are working to determine the structure and electronic structure of this molecule.  In addition to UV-Vis-NIR absorption spectroscopy at Barnard, we are developing collaborations with researchers at other institutions for investigations of MarR by electron paramagnetic resonance and Mössbauer spectroscopies and for analysis of metal content using inductively coupled plasma mass spectrometry.

Preliminary results from this project have been presented at two Gordon Research Conferences in the past year: the Metals in Biology GRC in Ventura, CA, in February 2010, and the Environmental Bioinorganic Chemistry GRC in Newport, RI, in June 2010. PRF funds paid part of the stipend of one undergraduate research student for Summer, 2010. One other undergraduate has also been working on the PRF-supported project; her stipend was provided by an institutional grant to Barnard College from Merck. In addition, these two students have performed undergraduate research for course credit during the academic year and one will write her senior thesis in chemistry on her work on this project.

Based in part on preliminary results from the PRF-funded research project, we have received major instrumentation and academic infrastructure grants from NSF that will support this work.  PRF support has been essential to our progress to date on this project.