While the iron-molybdenum cofactor (FeMoco) of nitrogenase, a constituent of the active site for nitrogen reduction, can be extracted into N-methylformamide (NMF) and pyrrollidinone, the inability to solubilize it in any other organic solvents has hampered further understanding of its structure and chemical properties. A method to solubilize FeMoco, prepared in N,N-dimethylformamide (DMF) with Bu4N+ as counterion [McLean, P. A., Wink, D. A., Chapman, S. K., Hickman, A. B., McKillop, D. M., & Orme-Johnson, W. H. (1989) Biochemistry (preceding paper in this issue)], in acetonitrile, acetone, methylene chloride, tetrahydrofuran, and benzene is reported. FeMoco evaporated to dryness in vacuo dissolves readily in good yield (55-100%) and with no significant loss in specific activity. In addition, FeMoco can be extracted directly into these solvents from MoFe protein bound to a DEAE-Sepharose column if the protein is pretreated with DMF. Methods have also been developed to extract fully active FeMoco into acetone and acetonitrile in the absence of any amide solvents (NMF or DMF). Extraction of FeMoco into acetone (30% yield) involves only pretreatment of column-bound protein with methanol, while extraction into acetonitrile (22% yield) requires pretreatment with methanol followed by THF. We conclude that the presence of a suitable soluble cation confers solubility to the cofactor in many common organic solvents and that the solubility of FeMoco in a given solvent may be independent of the ability of that solvent to extract the cofactor from column-bound protein.

A convenient and rapid method of obtaining the cofactor of nitrogenase (FeMoco) with a low and apparently limiting Fe/Mo ratio has been developed. FeMoco can be extracted from the MoFe protein bound to DEAE-cellulose. The cofactor is eluted in either N-methylformamide (NMF), N,N-dimethylformamide (DMF), or mixtures of these solvents by use of salts such as Et4NBr,Bu4NBr,Ph4PCl, and Ph4AsCl. The method is simple, is rapid (45 min), yields concentrated cofactor, and, unlike the original method [Shah, V. K., & Brill, W. J. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3249-3253] which requires anaerobic centrifugation, is easily scaled up. Furthermore, it gives yields of cofactor in excess of 70%. Its disadvantages are a high Fe:Mo ratio when DMF is the extracting solvent and a high salt concentration in the resultant FeMoco solution. These disadvantages are easily overcome by removing excess Fe by pretreating the cofactor with bipyridyl while still on the column. This gives Fe:Mo ratios of (6 +/- 1):1 (11 trials) with specific activities ranging from 170 to 220 nmol of C2H4/[min.(nmol of Mo)]. Chromatography on Sephadex LH-20 removes ca. 99% of the excess salt. The adsorption of MoFe protein to DEAE-cellulose seems to facilitate denaturation by organic solvents so that pretreatment of the protein with acid, used in earlier methods, is unnecessary. There is an apparent dependence on the charge density of the anion employed for elution of FeMoco bound to DEAE-cellulose, such that Cl- greater than Br- much greater than I-, PF6- is the order of effectiveness of the Bu4N+ salts of these anions.(ABSTRACT TRUNCATED AT 250 WORDS)

Previous Mössbauer and EPR studies of the MoFe protein (approximately 30 Fe and 2 Mo) of nitrogenase have revealed the presence of two unique clusters, namely, the P-clusters (presumably of the Fe4S4 type) and the molybdenum- and iron-containing cofactors (or M-clusters). Mössbauer components D (approximately 10-12 Fe) and Fe2+ (approximately 4 Fe) represent subsites of the P-clusters while component S (approximately 2 Fe) appeared to belong to a separate, unidentified cluster. In order to refine the analyses of Mössbauer spectra, we have constructed an isotopic hybrid of the Klebsiella pneumoniae protein which contains 57Fe-enriched P-clusters and 56Fe-enriched M-clusters. The highly resolved 57Fe Mössbauer spectra of this hybrid show that component S behaves spectroscopically like the P-cluster sites D and Fe2+ in oxidized and reduced MoFe protein. This suggests that S is a subset of the P-clusters rather than a different cluster type. The present study shows, for the first time, that the Debye-Waller factors of different P-cluster subsites have a different temperature dependence. Thus, the Fe2+/D absorption ratio is 4.0:10.0 at 4.2 K and 4.0:11.6 at 173 K. We propose that the reduced MoFe protein contains two pairs of P-clusters: one pair containing one Fe2+ and three D-sites and the other one Fe2+, two D, and one S-site. We have argued previously that the oxidized P-clusters occur in pairs as well.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTOn the difference between iron-molybdenum cofactor of wild-type and nifV- mutant molybdenum-iron proteins of Klebsiella pneumoniae: ENDOR, EXAFS, and EPR evidencePaul A. McLean, Anne E. True, Mark J. Nelson, Steve Chapman, Martin R. Godfrey, Boon K. Teo, W. H. Orme-Johnson, and Brian M. HoffmanCite this: J. Am. Chem. Soc. 1987, 109, 3, 943–945Publication Date (Print):February 1, 1987Publication History Published online1 May 2002Published inissue 1 February 1987https://pubs.acs.org/doi/10.1021/ja00237a075https://doi.org/10.1021/ja00237a075research-articleACS PublicationsRequest reuse permissionsArticle Views94Altmetric-Citations12LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts

When the iron-molybdenum cofactor (FeMoco) was extracted from the MoFe protein of nitrogenase from a nifV mutant of Klebsiella pneumoniae and combined with the FeMoco-deficient MoFe protein from a nifB mutant, the resultant MoFe protein exhibited the NifV phenotype, i.e. in combination with wild-type Fe protein it exhibited poor N2-fixation activity and its H2-evolution activity was inhibited by CO. These data provide strong evidence that FeMoco contains the active site of nitrogenase. The metal contents and e.p.r. properties of FeMoco from wild-type and nifV mutants of K. pneumoniae are very similar.

The MoFe protein of nitrogenase from Klebsiella pneumoniae nifV mutants, NifV- Kp1 protein, in combination with the Fe protein from wild-type cells, catalysed CO-sensitive H2 evolution, in contrast with the CO-insensitive reaction catalysed by the wild-type enzyme. The decrease in H2 production was accompanied by a stoicheiometric decrease in dithionite (reductant) utilization, implying that CO was not reduced. However, CO did not affect the rate of phosphate release from ATP. Therefore the ATP/2e ratio increased, indicating futile cycling of electrons between the Fe protein and the MoFe protein. The inhibition of H2 evolution by CO was partial; it increased from 40% at pH6.3 to 82% at pH 8.6. Inhibition at pH7.4 (maximum 73%) was half-maximal at 3.1 Pa (0.031 matm) CO. The pH optimum of the mutant enzyme was lower in the presence of CO. Steady-state kinetic analysis of acetylene reduction indicated that CO was a linear, intersecting, non-competitive inhibitor of acetylene reduction with Kii = 2.5 Pa and Kis = 9.5 Pa. This may indicate that a single high-affinity CO-binding site in the NifV- Kp1 protein can cause both partial inhibition of H2 evolution and total elimination of acetylene reduction. Various models to explain the data are discussed.

Klebsiella pneumoniae nitrogenase is a complex enzyme consisting of two component proteins, the tetrameric MoFe protein (Kp1) and the dimeric Fe protein (Kp2)1. Together these catalyse the ATP-dependent reduction of N2 and of several triple-bonded substrates such as C2H2, CN−, N−3 and CH3NC (refs 2, 3). In the absence of substrate, protons are reduced to H2 and all substrates compete with protons for reduction. Carbon monoxide inhibits all reductions except H2 evolution4. Seventeen genes are required for the synthesis of nitrogenase and expression of its activity in K. pneumoniae5–8, some of which are believed to be involved in processing of the nitrogenase proteins during synthesis9,10. Here we describe studies of the nitrogenase of bacteria mutated at one of these genes—nifV. These mutants produce nitrogenase which does not reduce N2 in vivo, although it does in vitro in conditions of high electron flux. We show that the defect is in the Kp1 component, and suggest that the nifV gene product modifies the Kp1 component so that N2 reduction will occur at the lower electron fluxes present in vivo, and that this might have been involved in the possible evolutionary development of the nitrogenase from a cyanide-detoxifying enzyme.