Mössbauer Study of the MoFe Protein of Nitrogenase from Azotobacter vinelandii Using Selective ⁵⁷Fe Enrichment of the M-Centers

Abstract

The molybdenum−iron (MoFe) protein of nitrogenase contains two unique metalloclusters called P-cluster [8Fe-7S] and M-center (FeMo cofactor, [7Fe-9S-Mo-homocitrate]). Using samples containing M-centers selectively enriched with 57Fe (57M56P), we have studied three M-center states with Mössbauer spectroscopy. The results are as follows. A detailed analysis of the Mössbauer spectra of the S = 3/2 state MN recorded in applied fields up to 8.0 T has revealed the features of the seventh Fe site which had eluded previous Mössbauer and ENDOR studies. This site has unusually small and anisotropic magnetic hyperfine interactions (A iso ≈ −4 MHz). Our studies have also revealed that the spectroscopic component previously labeled B 1 represents two equivalent Fe sites. Six of the M-center irons are trigonally coordinated to bridging sulfides; their unusual isomer shifts are discussed with particular reference to a trigonally coordinated Fe(II) thiolate complex synthesized by Power and co-workers (Inorg. Chem. 1995, 34, 1815−1822). The unusually low isomer shifts (δav = 0.41 mm/s) of MN suggest that the core of the M-center is (formally) best described as (Mo4+-3Fe3+-4Fe2+). The turnover complex MR is one electron further reduced than MN. While δav changes by 0.06 mm/s between the one-electron oxidized state MOX and MN, only a small change in δav, 0.02 mm/s, is observed between MN and MR. Moreover, spectra of the integer-spin state MR taken in strong applied magnetic fields are quite similar to those observed for MN, suggesting that the 7-Fe segment of the M-center has the same spin structure in both states. These observations suggest that the reduction MN → MR is associated mainly with the molybdenum site. In a preliminary experiment, we have also observed reduction of the M-cluster (ca. 40%) by irradiating a 57M56P sample at 77 K in a synchrotron X-ray beam. The radiolytically reduced state, MI, has integer electronic spin S ≥ 1, and its reduction appears to be centered on the iron components of the cluster.

Cleaving the N,N Triple Bond: The Transformation of Dinitrogen to Ammonia by Nitrogenases

“…This signal was later determined to arise from an integer-spin system (where S ! 2, with a ground-state non-Kramers doublet) [75], which is consistent with the prediction that the odd-number states of the MoFe protein cycle (i.e., M n states where n is an odd number) are likely to be non-Kramer systems [57,76].…”

“…The Mo atom, on the other hand, has a μ 6 -coordination, with homocitrate acting as a bidentate ligand through two oxygen atoms (one carboxyl group and one hydroxyl group) in addition to the ligation provided by three sulfides and one nitrogen atom of His α442 ( Figure 3). The homocitrate entity forms an extensive hydrogen-bonding network with the sulfides of the metal-sulfur core [4], and it is thought to be responsible for the overall negative charge of the FeMoco, which in turn is important for the insertion of FeMoco along a positively charged path into its binding site within the MoFe protein [56,57].…”

“…This approach has led to the identification of potential reaction intermediates [14,16] The presence of the M 4 state intermediate was first noticed in the Ile α70 -substituted MoFe protein, which had limited activities in the reduction of all substrates except protons, and later identified in the wild-type MoFe protein, which was present at low concentrations in the reaction mixture [69,70]. Kinetic and 57 Fe ENDOR spectroscopic studies suggest that the M 4 state intermediate is likely four electrons more reduced than the dithionite-reduced MoFe protein [67]. In the absence of any substrate, the MoFe protein in the M 4 state exhibits a novel S ¼1/2 EPR signal when freeze-quenched under slow turnover conditions [71].…”

Unification of reaction pathway and kinetic scheme for N 2 reduction catalyzed by nitrogenase

“…. ), "non-Kramers (NK)" states (9). Although NK-EPR signals at conventional microwave frequencies are well known for other enzymes (10,11), until now, no EPR signal from an integer-spin form of FeMo-co has been detected.…”

Biosynthesis of the Metalloclusters of Molybdenum Nitrogenase

“…The isolated FeMoco has been shown to be anionic (4), yet the proposed core charge of FeMoco in the resting state is ϩ1 or ϩ3 (37,55). The overall negative charge of the cofactor, therefore, is believed to originate from its endogenous homocitrate moiety, which is Ϫ4 if the -OH group is deprotonated.…”