Circular Dichroism and X-ray Spectroscopies of Azotobacter vinelandii Nitrogenase Iron Protein

Abstract

A detailed description of the function of MgATP hydrolysis in the nitrogenase substrate reduction mechanism has yet to emerge (1-5). The current model suggests that MgATP hydrolysis is coupled to electron transfer between the nitrogenase component proteins, the iron protein (Fe protein), 1 and the molybdenum-iron protein (MoFe protein). It is known that the Fe protein can bind two MgATP molecules and that this binding results in protein conformational changes essential to Fe protein docking to the MoFe protein (4 -7). The series of events following the docking of the Fe protein to the MoFe protein are not well understood, but it has been proposed that the hydrolysis of two MgATP molecules on the Fe protein precedes the transfer of an electron from the Fe protein [4Fe-4S] cluster to the MoFe protein for substrate reduction (8,9). Under optimal conditions, the dissociation of the oxidized Fe protein, with two bound MgADP molecules, is the rate-limiting step of the reaction (10, 11). Current models suggest that the above sequence of events are repeated until sufficient electrons have been transferred to the MoFe protein to carry out the reduction of the bound substrate.An understanding of any protein conformational changes induced in the nitrogenase Fe protein upon binding either MgATP or MgADP is essential to the elucidation of a detailed mechanism of nucleotide-bound conformational changes and the mechanism of coupling of MgATP hydrolysis to electron transfer and substrate reduction by nitrogenase. A significant consequence of nucleotide binding to the Fe protein are changes in the properties of its [4Fe-4S] cluster (4,5 (20 -22, 25-27). These studies showed that the CD of the Fe protein is measurable but weak in both oxidation states of the [4Fe-4S] cluster. Spectra from C. pasteurianum, Klebsiella pneumonia, and A. vinelandii were essentially identical, leading to the conclusion that the [4Fe-4S] cluster environment of these proteins is highly conserved. These studies further demonstrated that the oxidized Fe protein CD spectrum changed upon binding MgATP or MgADP. Both MgADP and MgATP binding were found to result in essentially the same CD spectrum (22,26,28).In order to define the function of MgATP binding and hydrolysis in the nitrogenase mechanism in conjunction with studies using site-specifically altered Fe proteins (6 -8, 29 2ϩ cluster in the Fe protein by dithionite was inferred from dithionite titration experiments using absorption, CD, and EPR spectroscopies. MATERIALS AND METHODSNitrogenase Proteins-Nitrogenase iron protein (Fe protein) and molybdenum-iron protein (MoFe protein) were purified anaerobically from A. vinelandii as described previously (29). Specific activities for the nitrogenase proteins were 2400 nmol of acetylene reduced⅐min Ϫ1 ⅐(mg of MoFe protein)Ϫ1 and 2200 nmol of acetylene reduced⅐minϪ1 . All proteins were homogeneous as determined by Coomassie staining of SDS-polyacrylamide gels (6). Protein concentrations were determined by a modified Biuret method with bovine serum albumin a...

Biosynthesis of (Bacterio)chlorophylls

“…For this purpose in nucleotide-dependent switch proteins minor conformational changes are induced by ATP binding and hydrolysis. Such alterations have been shown for the related nitrogenase protein NifH 2 by various three-dimensional structural analyses (16) as well as by CD spectroscopy in the 350 -600 nm range (24,47).…”

“…The spectral shape of ChlL 2 in the presence of nucleotides correlates well to CD spectra obtained in a related study for NifH 2 incubated with MgADP also showing positive deflections for the spectra visible at 340 and 480 nm. In contrast, ChlL 2 in the absence of nucleotides and ChlL 2 in the presence of ATP exhibited substantially different spectra when compared with nucleotidefree NifH 2 or NifH 2 in the presence of MgATP (24,47,48). From these experiments we concluded that ChlL 2 is a nucleotide-dependent switch protein triggering an ATPdependent conformational change that subsequently results in ternary complex formation.…”

“…For the related nitrogenase system it was demonstrated that such dynamic processes also have influence on the direct environment of the [4Fe-4S] cluster of NifH 2 (16,47,49) resulting in a more negative redox potential for this cluster (16,48). It was observed that upon nucleotide binding the geometry of the [4Fe-4S] cluster of NifH 2 was slightly altered (16).…”

Evidence for Electron Transfer-dependent Formation of a Nitrogenase Iron Protein-Molybdenum-Iron Protein Tight Complex

“…Upon hydrolysis of MgATP to MgADP and P i , the Fe protein is proposed to undergo additional protein conformational changes (2). This is deduced from the observation that the MgADP-bound state of the Fe protein exhibits distinct spectroscopic properties for the [4Fe-4S] cluster compared with the MgATP-bound state (18,20,23). Thus, it has been suggested that the conformational changes induced within the Fe protein in going from the MgATP-bound state to the MgADP-bound state represent a molecular switch, which could be coupled to electron transfer and dissociation of the protein-protein complex (2).…”

“…Full absorption spectra were recorded to confirm the oxidation state of the Fe protein and to determine the protein concentration. CD spectra were then recorded on an Aviv model 62DS spectropolarimeter and were base-line subtracted (18).…”