title: Interaction of acetylene and cyanide with the resting state of nitrogenase alpha-96-substituted MoFe proteins.
abstract: The nitrogenase MoFe protein contains the active site metallocluster called FeMo-cofactor [7Fe-9S-Mo-homocitrate] that exhibits an S = 3/2 EPR signal in the resting state. No interaction with FeMo-cofactor is detected when either substrates or inhibitors are incubated with MoFe protein in the resting state. Rather, the detection of such interactions requires the incubation of the MoFe protein together with its obligate electron donor, called the Fe protein, and MgATP under turnover conditions. This indicates that a more reduced state of the MoFe protein is required to accommodate substrate or inhibitor interaction. In the present work, substitution of an arginine residue (alpha-96(Arg)) located next to the active site FeMo-cofactor in the MoFe protein by leucine, glutamine, alanine, or histidine is found to result in MoFe proteins that can interact with acetylene or cyanide in the as-isolated, resting state without the need for the Fe protein, or MgATP. The dithionite-reduced, resting states of the alpha-96(Leu)-, alpha-96(Gln)-, alpha-96(Ala)-, or alpha-96(His)-substituted MoFe proteins show an S = 3/2 EPR signal (g = 4.26, 3.67, 2.00) similar to that assigned to FeMo-cofactor in the wild-type MoFe protein. However, in contrast to the wild-type MoFe protein, the alpha-96-substituted MoFe proteins all exhibit changes in their EPR spectra upon incubation with acetylene or cyanide. The alpha-96(Leu)-substituted MoFe protein was representative of the other alpha-96-substituted MoFe proteins examined. The incubation of acetylene with the alpha-96(Leu) MoFe protein decreased the intensity of the normal FeMo-cofactor signal with the appearance of a new EPR signal having inflections at g = 4.50 and 3.50. Incubation of cyanide with the alpha-96(Leu) MoFe protein also decreased the FeMo-cofactor EPR signal with concomitant appearance of a new EPR signal having an inflection at g = 4.06. The acetylene- and cyanide-dependent EPR signals observed for the alpha-96(Leu)-substituted MoFe protein were found to follow Curie law 1/T dependence, consistent with a ground-state transition as observed for FeMo-cofactor. The microwave power dependence of the EPR signal intensity is shifted to higher power for the acetylene- and cyanide-dependent signals, consistent with a change in the relaxation properties of the spin system of FeMo-cofactor. Finally, the alpha-96(Leu)-substituted MoFe protein incubated with (13)C-labeled cyanide displays a (13)C ENDOR signal with an isotropic hyperfine coupling of 0.42 MHz in Q-band Mims pulsed ENDOR spectra. This indicates the existence of some spin density on the cyanide, and thus suggests that the new component of the cyanide-dependent EPR signals arise from the direct bonding of cyanide to the FeMo-cofactor. These data indicate that both acetylene and cyanide are able to interact with FeMo-cofactor contained within the alpha-96-substituted MoFe proteins in the resting state. These results support a model where effective interaction of substrates or inhibitors with FeMo-cofactor occurs as a consequence of both increased reactivity and accessibility of FeMo-cofactor under turnover conditions. We suggest that, for the wild-type MoFe protein, the alpha-96(Arg) side chain acts as a gatekeeper, moving during turnover in order to permit accessibility of acetylene or cyanide to a specific [4Fe-4S] face of FeMo-cofactor.
authors.affiliation: Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322, USA.
authors.forename: P M
authors.initials: PM
authors.lastname: Benton
authors.forename: S M
authors.initials: SM
authors.lastname: Mayer
authors.forename: J
authors.initials: J
authors.lastname: Shao
authors.forename: B M
authors.initials: BM
authors.lastname: Hoffman
authors.forename: D R
authors.initials: DR
authors.lastname: Dean
authors.forename: L C
authors.initials: LC
authors.lastname: Seefeldt
mesh_terms: D000114:Acetylene / Q000378:metabolism*; D019943:Amino Acid Substitution; D001120:Arginine / Q000378:metabolism; D016948:Azotobacter vinelandii / Q000201:enzymology; D001665:Binding Sites; D002248:Carbon Monoxide / Q000737:chemistry; D020134:Catalytic Domain; D003486:Cyanides / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004791:Enzyme Inhibitors / Q000737:chemistry; D005973:Glutamine / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D007930:Leucine / Q000378:metabolism; D008983:Molybdoferredoxin / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D009591:Nitrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D003486:Cyanides; D004791:Enzyme Inhibitors; D008983:Molybdoferredoxin; D005973:Glutamine; D006639:Histidine; D002248:Carbon Monoxide; D001120:Arginine; D009591:Nitrogenase; D007930:Leucine; D000114:Acetylene