William H. Orme-Johnson

Reversible conversion of coenzyme F420 to the 8-OH-AMP and 8-OH-GMP esters, F390-A and F390-G, on oxygen exposure and reestablishment of anaerobiosis in Methanobacterium thermoautotrophicum

Intracellular levels of F390 (AMP and GMP adducts of the 5-deazaflavin cofactor F420) in Methanobacterium thermoautotrophicum were analysed after gasing fermenter cultures with several consecutive cycles of substrate gas and gas mixtures containing 5% oxygen. No F390 was detected in growing cells, hydrogen starved cells and CO2 starved cells prior to O2 contamination. Also, no F390 was found in hydrogen depleted cells after O2 treatment. Exposure of exponentially growing cells and CO2 starved cells to oxygen lead to the formation of F390 species; the increase in the detected amount of F390 was coupled to a decrease of the F420 level. As soon as anaerobiosis was reestablished F390 cofactors were degraded and growth proceeded. Independent of the physiological condition of Methanobacterium thermoautotrophicum methanopterin was formed upon O2 exposure. After normal growth conditions were restored the level of detected methanopterin decreased again.

The nature of amide ligation to the metal sites of FeMoco

Abstract The nature of the ligation of N-methylformamide (NMF) to the metal sites of FeMoco has been investigated. The IR spectrum of FeMoco in NMF shows an intense band at 1600 cm−1 which we assign to the v(CO) stretch of ligated NMF. This frequency is characteristic of N-deprotonated amide ligands, indicating that NMF ligation to FeMoco is predominantly through nitrogen. Hence NMF behaves as an anionic ligand. The importance of the amide functionality for FeMoco extraction has been further examined and a new extracting agent, 2-pyrrolidinone, has been employed. We hypothesize that, in the MoFe-protein, the primary coordination of the cofactor is via deprotonated backbone amide ligands.

A novel vanadium(V) homocitrate complex: synthesis, structure, and biological relevance of [K2(H2O)5][(VO2)2(R,S-homocitrate)2]·H2O

Abstract Initial investigations into the possible roles of homocitric acid in the biosynthesis and function of the active site cofactor of nitrogenase resulted in the isolation and characterization of the dinuclear vanadium(V) species [K2(H2O)5][(VO2)2(R,S-C7H8O7)2]·H2O ( 1). Complex 1 represents the first synthetic structurally characterized transition metal homocitrate complex and may represent an early mobilized precursor in the biosynthesis of VFeco. Compound 1 was characterized by a variety of physical methods, including X-ray crystallography. Crystal data: space group P * (#2), with a = 10.292 (3) Å, b = 16.663 (3) Å, c = 8.343 (1) Å, α = 95.93 (1)°, β = 105.74 (2)°, γ = 90.86 (2)°, V = 1386 (1) Å3, and Z = 2. The homocitrate ligand is coordinated to the vanadium(V) atoms in a bidentate fashion via the deprotonated bridging hydroxyl group and a carboxylate donor. This unique coordination mode accurately mimics the coordination of homocitrate to the cofactor of nitrogenase.

THE EFFECT OF CALPROTECTIN ON THE NUCLEATION AND GROWTH OF STRUVITE CRYSTALS AS ASSAYED BY LIGHT MICROSCOPY IN REAL-TIME

PURPOSE To use light microscopy to observe the urease-induced growth of struvite crystals in real-time, and to compare the effects of various proteins on that growth. MATERIALS AND METHODS Artificial urine, with and without citrate, and a minimal urine solution containing only urea and the components of struvite and apatite were incubated with urease and test proteins in the depressions of culture slides. The number and size of rectangular and X-shaped struvite crystals were recorded using a low-power phase contrast microscope. RESULTS The formation of crystalline struvite appears to occur after the formation of an amorphous calcium- and magnesium-containing phase. The extent of this amorphous phase is dependent on the presence of calcium and citrate, both of which strongly promote its formation over the crystalline phase. alpha-globulin, gamma-globulin and chymotrypsin inhibitor all result in the same amount of crystalline struvite as bovine serum albumin which is used as a control. Calprotectin, on the other hand, causes consistent and significant reductions in the number and size of struvite crystals under a wide range of conditions. No changes in the morphology of the struvite crystals were observed. CONCLUSIONS Calprotectin, the dominant protein of infection stone matrix, has distinctive properties which affect the formation and growth of struvite crystals. The presence of citrate in synthetic urine dramatically reduces the number of struvite crystals observed. The present method for observing the effects of putative infection stone inhibitors appears to have merit.

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.

Increased amplitudes of electron spin echoes by phase shifted excitation

Excitation by two 90° pulses in phase quadrature lengthens the phase memory time of electron spin echoes. This prolongation enhances the accuracy with which modulations arising from hyperfine interactions can be measured in relatively concentrated spin systems. Comparison of these echoes with ones produced normally (without the phase shift) allow one to measure selectively the electron–electron spin coupling tensor.

Kinetic, spectroscopic, and structural (extended X-ray absorption fine structure) studies on the type 1 blue copper protein umecyanin

The effects of pH variations on the visible spectrum (pKa 9.46 ± 0.04), e.s.r. spectrum, extended X-ray absorption fine structure (EXAFS), and on the reactivity of the blue copper protein umecyanin have been investigated. The electron-transfer reactions of umecyanin have been studied with the complexes [Co(dipic)2]–(dipic = dipicolinate) and [Co(C2O4)3]3– as oxidants for UCu(I), and [Ru(NH3)5(py)]2+(py = pyridine) as a reductant for UCu(II), at 25 °C, I= 0.10 M (NaCl). The oxidation of umecyanin with [Fe(CN)6]3– is extremely rapid, consequently a limited study was carried out at a lower temperature. The oxidation of the protein by [Co(phen)3]3+(phen = 1,10-phenanthroline) was also studied under these conditions for comparison. Of interest are the pKa values observed for the oxidation of umecyanin by [Co(C2O4)3]3–(pKa 9.68 ± 0.08) and the reduction of umecyanin by [Ru(NH3)5(py)]2+(pKa 9.50 ± 0.07). The low temperature (20 K)X-band e.s.r. spectrum of oxidised umecyanin (g‖= 2.32, g⊥= 2.06) shows little variation over the pH range 7.5–10.5. The Cu K-edge EXAFS of umecyanin in both oxidation states and at pH 7.5 and 10.5 were obtained. Analysis of data from the oxidised protein shows the Cu atom has two nitrogen ligands at 1.99 ± 0.03 A and one sulphur at 2.13 ± 0.02 A. In the reduced protein the bond lengths increase to 2.03 ± 0.03 A and 2.21 ± 0.02 A respectively. There was no detectable difference in co-ordination number or bond lengths at the different pH values, and no fourth ligand could be detected by the EXAFS technique. The results from these various studies are discussed in terms of changes at or near the active site of umecyanin.

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.