Mossbauer study of the MoFe protein of nitrogenase from Azotobacter vinelandii using selective 57Fe enrichment of the M-centers

The molybdenum-iron (MoFe) protein of nitrogenase contains two unique metallocrusters called P-cluster [8Fe-7S] and M-center (FeMo cofactor, [7Fe-9S-Mo-homocitrate]). Using samples containing M-centers selectively enriched with Fe-57 ((MP)-M-57-P-56), we have studied three M-center states with Mossbauer spectroscopy. The results are as follows. A detailed analysis of the Mossbauer 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 Mossbauer and ENDOR studies. This site has unusually small and anisotropic magnetic hyperfine interactions (A(iso) approximate to -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 (delta(av) = 0.41 mm/s) of M-N suggest that the core of the M-center is (formally) best described as (Mo4+-3Fe(3+)-4Fe(2+)). The turnover complex MR is one electron further reduced than MN. While delta(av) changes by 0.06 mm/s between the one-electron oxidized state M-OX and M-N, only a small change in delta(av), 0.02 mm/s, is observed between M-N and M-R. 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 M-N --> M-R 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 (57)M(56)p Sample at 77 K in a synchrotron X-ray beam. The radiolytically reduced state, M-I, has integer electronic spin S greater than or equal to 1, and its reduction appears to be centered on the iron components of the cluster.