title: Mössbauer characterization of the metal clusters in Azotobacter vinelandii nitrogenase VFe protein.
abstract: The VFe protein of alternative nitrogenase, isolated from Azotobacter vinelandii, strain LS15 and designated as Av1', has been investigated by Mössbauer spectroscopy. The Mössbauer spectrum of the dithionite-reduced Av1', recorded at 4.2 K with a 60-millitesla magnetic field applied parallel to the gamma-beam, is a superposition of three spectral components: 1) a complex spectrum (the M component) with magnetic hyperfine structures attributed to the paramagnetic FeV cofactor, 2) a component (the P component) consisting of three quadrupole doublets identifiable as the Fe2+, D, and S doublets similar to those observed for the P cluster pairs in MoFe proteins, and 3) a minor (4% of total absorption) quadrupole doublet attributable to adventitiously bound iron. The observed 4.2-K parameters for the Fe2+ (delta EQ = 2.99 mm/s and delta = 0.64 mm/s), D (delta EQ = 0.75 mm/s and delta = 0.63 mm/s), and S (delta EQ = 1.2 mm/s and delta = 0.65 mm/s) iron sites and their temperature dependence are very similar to those observed for the P cluster pairs in the conventional MoFe proteins. Similar to those of the MoFe protein, strong field spectra indicate that these doublets are associated with a diamagnetic system. Their percent absorption intensities (Fe(2+)/D/S = 13.0:32.2:6.8) determined at 4.2 K after the removal of the contributions from the adventitiously bound iron are comparable to those of the P cluster pairs in MoFe proteins. These observations established that Av1' also contains P cluster pairs that are identical, in both composition and quantity, to those of the MoFe proteins; i.e. each molecule contains two P cluster pairs and each pair is formed by two Fe2+, five D, and one S iron sites. Considering that 52% absorption of the P component corresponding to two 8Fe clusters, the remaining 48% absorption determined for the M component is consistent with two 7Fe-containing FeV cofactors/molecule of Av1'. The fact that both P cluster pairs are found in the diamagnetic states implies that the S = 3/2 and S = 1/2 signals detected in earlier EPR measurements are associated with the FeV cofactor and suggests a spin mixture for the FeV cofactor in the dithionite-reduced Av1'.
authors.affiliation: Department of Physics, Rollins Research Center, Emory University, Atlanta, Georgia 30322.
authors.forename: N
authors.initials: N
authors.lastname: Ravi
authors.forename: V
authors.initials: V
authors.lastname: Moore
authors.forename: S G
authors.initials: SG
authors.lastname: Lloyd
authors.forename: B J
authors.initials: BJ
authors.lastname: Hales
authors.forename: B H
authors.initials: BH
authors.lastname: Huynh
mesh_terms: D016948:Azotobacter vinelandii / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry*; D007501:Iron / Q000737:chemistry*; D009591:Nitrogenase / Q000737:chemistry*; D009942:Organometallic Compounds / Q000737:chemistry; D015204:Spectroscopy, Mossbauer; D014639:Vanadium / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D009942:Organometallic Compounds; D014639:Vanadium; D007501:Iron; D009591:Nitrogenase