Probing the MgATP-Bound Conformation of the Nitrogenase Fe Protein by Solution Small-Angle X-ray Scattering

Abstract

The MgATP-bound conformation of the Fe protein of nitrogenase from Azotobacter vinelandii has been examined in solution by small angle x-ray scattering (SAXS) and compared to existing crystallographically characterized Fe protein conformations. The results of the analysis of the crystal structure of an Fe protein variant with a Switch II single amino acid deletion recently suggested that the MgATP-bound state of the Fe protein may exist in a conformation that involves a large-scale reorientation of the dimer subunits resulting in an overall elongated structure relative to the more globular structure of the MgADP-bound state. It was hypothesized that the Fe protein variant may be a conformational mimic of the MgATP-bound state of the native Fe protein based largely on the observation that the spectroscopic properties of the [4Fe-4S] cluster of the variant mimicked in part the spectroscopic signatures of the native nitrogenase Fe protein in the MgATPbound state. In the present work, SAXS studies reveal that the large scale conformational differences between the native Fe protein and the variant observed by x-ray crystallography are also observed in solution. In addition, comparison of the SAXS curves of the Fe protein nucleotide-bound states to the nucleotide-free states indicates that the conformation of the MgATP-bound state in solution does not resemble the structure of the variant as initially proposed, but rather, at the resolution of this experiment, it resembles the structure of the nucleotide-free state. These results provide insights into the Fe protein conformations that define the role of MgATP in nitrogenase catalysis.

Mechanism of Mo-Dependent Nitrogenase

“…Given that the MgATP binding sites within the Fe protein are located some distance away (> 10 Å) from the MoFe protein docking interface (13), this situation demands protein conformational changes induced within the Fe protein that activate MgATP hydrolysis (28, 31). Two specific “switch” regions (stretches of amino acids) within the Fe protein have been proposed to function to communicate between the docking interface and the MgATP binding site, resulting in movement of key catalytic residues that result in activation of nucleotide hydrolysis (7, 15, 20, 22–24, 38, 39). Although broad strokes of this process have been defined, details of the mechanism remain unresolved.…”

Mechanism of Mo-Dependent Nitrogenase

“…Given that the MgATP binding sites within the Fe protein are located some distance away (> 10 Å) from the MoFe protein docking interface (13), this situation demands protein conformational changes induced within the Fe protein that activate MgATP hydrolysis (28,31). Two specific "switch" regions (stretches of amino acids) within the Fe protein have been proposed to function to communicate between the docking interface and the MgATP binding site, resulting in movement of key catalytic residues that result in activation of nucleotide hydrolysis (7,15,20,(22)(23)(24)38,39). Although broad strokes of this process have been defined, details of the mechanism remain unresolved.…”

“…The enzyme is composed of two-component metalloproteins called the iron (Fe) protein (also called dinitrogenase reductase or component II) and the molybdenum-iron (MoFe) protein (also called dinitrogenase or component I) ( Figure 1). The structures of the two nitrogenase component proteins (individually and in complex with each other), have been determined by X-ray crystallography (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). These structures have provided detailed models of the three metal clusters contained in nitrogenase.…”

Mechanism of Nitrogen Fixation by Nitrogenase: The Next Stage

“… 90 − 98 Recent studies have begun to shed light on the order of events during the catalytic cycle, 99 − 103 including the nature of electron transfer between the metal clusters 62 , 104 − 111 and the roles of ATP binding and hydrolysis in these processes. 55 , 68 , 99 , 112 − 121 Considerable progress has been made in the application of theoretical methods to various aspects of the nitrogenase mechanism. 122 − 140 Finally, progress has been made in expanding in the substrates of nitrogenases 93 , 141 − 149 to include CO 95 , 96 , 98 , 150 , 151 and CO 2 .…”