Mössbauer Study of the MoFe Protein of Nitrogenase from <i>Azotobacter vinelandii</i> 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 (Aiso ≈ −4 MHz). Our studies have also revealed that the spectroscopic component previously labeled B1 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.