Carol A. Gormal

Crystallographic analysis of the MoFe protein of nitrogenase from a nifV mutant of Klebsiella pneumoniae identifies citrate as a ligand to the molybdenum of iron molybdenum cofactor (FeMoco).

The x-ray crystal structure of NifV− Klebsiella pneumoniae nitrogenase MoFe protein (NifV− Kp1) has been determined and refined to a resolution of 1.9 Å. This is the first structure for a nitrogenase MoFe protein with an altered cofactor. Moreover, it is the first direct evidence that the organic acid citrate is not just present, but replaces homocitrate as a ligand to the molybdenum atom of the iron molybdenum cofactor (FeMoco). Subsequent refinement of the structure revealed that the citrate was present at reduced occupancy.

EXPLORING THE REACTIVITY OF THE ISOLATED IRON-MOLYBDENUM COFACTOR OF NITROGENASE

Abstract There is strong evidence that the iron-molybdenum cofactor (FeMoco) of nitrogenase forms part of the enzyme’s active site. FeMoco, a MoFe7S9·homocitrate cluster, can be extracted intact from the enzyme into N-methylformamide solution but is reported to be inactive in substrate reduction unless powerful reductants are used and then only acetylene and cyclopropene reductions have been observed. The literature on the catalytic and substrate binding reactivities of extracted FeMoco is reviewed and new data on electrocatalytic hydrogen evolution presented. A comparison of the ligand binding properties of FeMoco from the wild-type and a NifV− mutant enzyme, which has citrate in place of R-homocitrate, is presented. These data are interpreted in terms of their significance for enzyme turnover and of the obligate requirement for R-homocitrate for dinitrogen reduction.

X-ray absorption spectroscopic studies of the binding of ligands to FeMoco of nitrogenase from Klebsiella pneumoniae

The binding of ligands to the iron-molybdenum cofactor (FeMoco) from Klebsiella pneumoniae nitrogenase has been studied by XANES and EXAFS at the Fe. Mo and Se k-edges and by EPR. Ligands investigated include the anions derived from thiophenol, 2-bromophenylthiol, phenylselenol, a trithiolate ligand, 1, and the cyanate and thiocyanate anions. Evidence has been obtained directly that phenylselenol, and indirectly that thiophenol, 2-bromophenylthiol and ligand, 1, are bound by iron. For FeMoco plus phenylselenol, an iron-selenium distance of 2.36 A was determined from the Fe k-edge data and 2.38 A from the Se k-edge data. The results suggest that one selenol (and by analogy thiophenol and 2-bromophenylthiol) is bound in a non-bridging mode by FeMoco. Binding of ligand 1 led to a large splitting of the Fe shell at the Mo k-edge. At the Fe k-edge, binding of ligand 1 led to large decreases in the intensity of the iron-iron contacts. No evidence was found for cyanate or thiocyanate binding to Fe.

The isolated iron–molybdenum cofactor of nitrogenase catalyses hydrogen evolution at high potential

The isolated cofactor of nitrogenase FeMoco catalyses hydrogen evolution at the high potential associated with the FeMocoox/semi-red couple (E° = –280 mV vs. NHE , C6F5S– ligated form); analysis of the current–potential dependence of the catalysis suggests a mechanism involving rate-determining loss of H2 from an FeMoco(H)2red intermediate (k = 3 s–1); the relatively slow kinetics of this step may be related to an obligatory role for hydridic intermediates in substrate reductions by nitrogenase.

The isolated iron–molybdenum cofactor of nitrogenase binds carbon monoxide upon electrochemically accessing reduced states

The first spectroscopic evidence for the binding of a small gaseous molecule to the isolated iron molybdenum cofactor of nitrogenase (FeMoco) is presented: FTIR spectroelectrochemistry in a thin-layer cell shows that reduced FeMoco binds carbon monoxide and gives rise to ν(CO) stretches that are close to those observed during turnover of the enzyme.

A new approach to identifying substrate binding sites on isolated FeMo-cofactor of nitrogenase

A kinetic method is described which is capable of detecting the binding of molecules and ions such as CN - , N 3 - , Bu t NC, imidazole and H + to the isolated FeMo-cofactor of nitrogenase, and indicates where on the cofactor these species bind.

Structure of Klebsiella pneumoniae Nitrogenase

The 3D crystallographic structure of the nitrogenase MoFe protein was first determined for the protein from Azotobacter vinelandii (Av1) at 2.8 A resolution and identified two unique metal-sulphur clusters viz the FeMoco centres and the P clusters (Kim, Rees 1992). This structure was later refined to 2.2 A resolution (Chan et al, 1993). Analysis of the structure of the MoFe protein from Clostridium pasteurianum (Cpl) was consistent with the formulation of FeMoco as MoFe7S9. homocitrate but differed in the interpretation of the structure of the P clusters (Bolin et al, 1993). Rees’s group reported that the P clusters consisted of two Fe4S4 clusters bonded through a disulphide bridge at one corner whereas Bolin suggested that the two Fe4S4 cubanes shared a single sulphur atom at the corner to produce an Fe8S7 cluster. (Fig 1)