EXAFS STUDIES OF FEMO-COFACTOR AND MOFE PROTEIN - DIRECT EVIDENCE FORTHE LONG-RANGE MO-FE-FE INTERACTION AND CYANIDE BINDING TO THE MO IN FEMO-COFACTOR

The biological reduction of dinitrogen to ammonia by the nitrogenase s ystem requires the MoFe protein, which contains two iron-molybdenum co factors (FeMoco) and two Fe-S P-clusters, and the Fe protein, which is the electron donor in catalysis. The two FeMo-cofactors are the likel y sites of substrate binding and reduction, and knowledge of their det ailed structure and function is central to understanding the chemistry of this complex enzyme system. Recent crystal structure studies of th e MoFe protein are providing remarkable details about the MoFe protein , but questions about the structure of isolated FeMoco and reactivity of the FeMoco site remain unanswered. We report herein a new series of Mo-K edge EXAFS studies of highly concentrated isolated FeMoco, isola ted FeMoco plus CN-, and the MoFe protein. Very high quality data has been obtained over a wide k-range through improved experimental techni ques. In addition, new EXAFS analysis methodology (called GNXAS) based on multiple scattering formalism with further enhancements has been u sed to analyze the data. Several important results have emerged: for t he first time a second shell of Fe atoms at approximately 5.1 angstrom from the Mo is clearly present in the EXAFS for both FeMoco and the M oFe protein. This provides direct evidence for the ''intact'' nature o f extracted FeMoco and demonstrates the ability to detect and analyze such long-range absorber-scatter interactions. Second, the EXAFS resul ts give very accurate metrical details of the FeMoco sites, and these differ from those of FeMoco from the X-ray crystal structure of the Mo Fe protein at its current level of refinement. Finally, using the GNXA S analysis method, it is shown that added CN- coordinates to Mo in iso lated FeMoco. These results further define accurate metrical details o f FeMoco within and outside of the protein and provide the methodologi cal basis for further investigations of chemical reactivity.