Paul A. McLean

Effects of substrates (methyl isocyanide, C2H2) and inhibitor (CO) on resting-state wild-type and NifV(-)Klebsiella pneumoniae MoFe proteins.

We report the use of electron nuclear double resonance (ENDOR) spectroscopy to examine how the metal sites in the FeMo-cofactor cluster of the resting nitrogenase MoFe protein respond to addition of the substrates acetylene and methyl isocyanide and the inhibitor carbon monoxide. 1H, 57Fe and 95Mo ENDOR measurements were performed on the wild-type and the NifV(-)proteins from Klebsiella pneumoniae. Among the molecules tested, only the addition of acetylene to either protein induced widespread changes in the 57Fe ENDOR spectra. Acetylene also induced increases in intensity from unresolved protons in the proton ENDOR spectra. Thus we conclude that acetylene may bind to the resting-state MoFe protein to perturb the FeMo-cofactor environment. On the other hand, the present results show that methyl isocyanide and carbon monoxide do not substantially alter the FeMo cofactors geometric and electronic structures. We interpret this as lack of interaction between those two molecules and the FeMo cofactor in the resting state MoFe protein. Thus, although it is generally accepted that substrates or inhibitors bind to the FeMo-cofactor only under turnover condition, this work provides evidence that at least one substrate can perturb the active site of nitrogenase under non-catalytic conditions.

Overproduction of nif KD and nif SUX from Klebsiella Pneumoniae

The molybdenum-iron protein and the iron protein from Klebsiella pneumoniae catalyze the reduction of dinitrogen to ammonia. The MoFe-protein, believed to harbour the site(s) for dinitrogen binding, contains 2 bound molybdenum, 30–32 irons and 28 acid-labile sulfurs. These metals are arranged in 3 types of clusters: 2 M-centers each consisting of 8 irons, 8 acid-labile sulfurs and 1 molybdenum atom. This FeMo-cofactor can be extracted into N-methyl formamide; 4[4Fe-4s] clusters called P-clusters; and 2 residual irons termed the S-cluster(s). A total of 16 genes have been implicated in regulation and synthesis of both introgenase proteins. The genes coding for the structural polypeptides of iron- and molybdenum- iron proteins are contained in one operon (nif HKDY). The nif H promoter is not only regulated by the activator nif A and the repressor nif L but also reguires a fully matured MoFe-protein and/or Fe-protein for maximal expression. The products of 4–5 genes (nif B, N. E, V and C) are involved in insertion of the FeMo-cofactor into the MoFe-protein. Whether the remaining metal clusters, which are believed to be inserted prior to the FeMo-cofactor, are matured by an enzymatic process remains to be determined. There are several genes, notably nif SUX, whose functions are yet obscure; some of these may participate in P-cluster and S-cluster insertion.

Construction of Plasmids that Overproduce nif Regulatory Proteins

The functions and interactions of the nif regulatory proteins, nif A and nif L are not well understood. Our objective is to purify, study and further characterize these proteins. The expression of these proteins in vivo, however, is controlled by the glutamine synthetase regulatory (gln) system. We sought a way to produce nif A and nif L in a gln-free background since the regulatory properties of both types of proteins (e.g. gln G and nif A) have been shown to be similar. This could present problems later in assays and purification.

Construction of Recombinant Plasmids which Overproduce the nif B, Q, N, E, & X Gene Products

The aim of this work is to determine the structure and synthetic pathway of the iron-molybdenum cofactor (FeMo-co) of nitrogenase. In order to study the functions of the genes implicated In synthesis and/or insertion of FeMo-co (nif B, nif E, nif N, nif N, nif V and possibly nif Q and nif X)1,2,3, we are currently constructing recombinant plasmids which overproduce the proteins coded for by these genes and, subsequently, will purify these gene products. Overproduction is necessary because there are no known assays for these gene products, and so their purification will have to be monitored by polyacrylamide gel electro phoresis, therefore requiring easy visibility of protein bands.