Structure determination and refinement at 2.2 angstroms of nitrogenase molybdenum iron-protein from Clostridium pasteurianum

Nino Campobasso, Purdue University

Abstract

Nitrogenase catalyzes the reduction of dinitrogen through the activity of a complex of two easily separated components known as the MoFe-protein and the Fe-protein. The MoFe-protein is the site of N$\sb2$ binding and reduction, whereas the Fe-protein provides electrons and binds ATP. MoFe-protein is a large (M$\sb{\rm r}$ = 230,000), $\alpha\sb2\beta\sb2$, tetrameric enzyme that contains two copies each of two unusual metal clusters known as the FeMo-cofactor and the P-cluster. The crystal structure of MoFe-protein from Clostridium pasteurianum has been determined to 2.2A resolution by a combination of phasing methods including multiwavelength anomalous diffraction, isomorphous replacement, and phase extension involving both solvent flattening and molecular averaging. Both rotating anode and synchrotron (SSRL, CHESS, and Photon Factory) sources were used for diffraction experiments at a variety of wavelengths including 1.0A, 1.54A, 1.74A, and 1.79A. An atomic model of MoFe-protein, with its bound metal-sulfur clusters, 2 Mg$\sp{2+}$ ions, and 1,004 water molecules, has been refined by least-squares techniques against crystallographic data and stereochemical restraints. The refined model has an R-factor of 15.1% based on all measured crystallographic data between 25A and 2.2A. The protein model has root mean-square deviations from ideal bond distances and angles of 0.006A and 1.67$\sp\circ$, respectively. The crystalloeraphic results are interpreted with respect to the chemical mechanism of N$\sb2$ reduction and intramolecular electron transfer.

Degree

Ph.D.

Advisors

Bolin, Purdue University.

Subject Area

Biochemistry|Biophysics

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