Barry E. Smith

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.

Structures of a blue-copper nitrite reductase and its substrate-bound complex.

Copper-containing nitrite reductases (NiRs) have been conveniently subdivided into blue and green NiRs which are thought to be redox partners of azurins and pseudo-azurins, respectively. Crystal structures of two green NiRs have recently been determined. Alcaligenes xylosoxidans has been shown to have a blue-copper nitrite reductase (AxNiR) and two azurins with 67% homology both of which donate electrons to it effectively. The first crystal structure of a blue NiR (AxNiR) in its oxidized and nitrite-bound forms, with particular emphasis to the Cu sites, is presented. The Cu-Smet distance is the same as those in the green NiRs. Thus, the length of this interaction is unlikely to be responsible for differences in colour. Crystallographic data presented here taken together with structural data of other single Cu type-1 proteins and their mutants suggest that the displacement of Cu from the strong ligand plane is perhaps the cause for the differences in colour observed for otherwise classical blue Cu centre. Nitrite is observed binding to the catalytic Cu in a bidentate fashion displacing the water molecule, offering a neat rationalization for the XAFS observation that the type-2 Cu-ligand distances increase on nitrite binding as a result of increased coordination. These results are discussed in terms of enzyme mechanism.

The substrate-binding site in Cu nitrite reductase and its similarity to Zn carbonic anhydrase.

Here we investigate the structure of the two types of copper site in nitrite reductase from Alcaligenes xylosoxidans, the molecular organisation of the enzyme when the type-2 copper is absent, and its mode of substrate binding. X-ray absorption studies provide evidence for a fourth ligand at the type-2 Cu, that substrate binds to this site and indicates that this binding does not change the type-1 Cu centre. The substrate replaces a putative water ligand and is accommodated by a lengthening of the Cu–histidine bond by approximately 0.08 Å. Modelling suggests a similarity between this unusual type-2 Cu site and the Zn site in carbonic anhydrase and that nitrite is anchored by hydrogen bonds to an unligated histidine present in the type-2 Cu cavity.

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.

Comparing crystallographic and solution structures of nitrogenase complexes

A low-resolution structure from X-ray scattering data of Kp1•(ADP•AlF-_4•Kp2)2 predicted a significant change in the iron protein (Kp2) upon complex formation. This has been subsequently confirmed by the crystallographic structure of the complex in the Av system. New scattering results are provided to demonstrate the similarity of this complex in the two species.

XAFS studies of nitrogenase: the MoFe and VFe proteins and the use of crystallographic coordinates in three‐dimensional EXAFS data analysis. Erratum

This paper reports a three-dimensional EXAFS refinement of the Mo coordination sphere of the FeMoco cluster of the dithionite-reduced MoFe protein from Klebsiella pneumoniae nitrogenase (Kp1) using the 1.6 A-resolution crystallographic coordinates. At this resolution, the positions of the heavy (Fe and S) atoms of the cluster are well determined and there is excellent agreement between the crystallographic and EXAFS models. However, the lighter homocitrate and histidine ligands are poorly determined in the crystal structure, and it is shown that the application of EXAFS-derived distance restraints during the early stages of crystallographic refinement provides a means of substantially improving (by approximately 0.1 A) the final crystallographic model. The consistency of the EXAFS analysis with the crystallographic information in this case justifies applications of EXAFS to cases where protein crystal structures are absent. Thus, the VFe protein of V-nitrogenase has been shown by EXAFS to possess a V-atom site catalytically similar to the well characterized MoFe-nitrogenases, with V replacing Mo.

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 nof molecules and ions such as CN n - n, nN n 3 n n - n, Bu n t nNC, imidazole and nH n + n to the isolated FeMo-cofactor of nitrogenase, and indicates nwhere on the cofactor these species bind.