Interaction of acetylene and cyanide with the resting state of nitrogenasealpha-96-substituted MoFe proteins

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 ei ther substrates or inhibitors are incubated with MoFe protein in the restin g 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 res idue (alpha -96(Arg)) located next to the active site FeMo-cofactor in the MoFe protein by leucine, glutamine, alanine, or histidine is found to resul t in MoFe proteins that can interact with acetylene or cyanide in the as-is olated, resting state without the need for the Fe protein, or MgATP. The di thionite-reduced, resting states of the alpha -96(Leu)-, alpha -96(Gln)-, a lpha -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-cofacto r 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 th eir EPR spectra upon incubation with acetylene or cyanide. The alpha -96(Le u)-substituted MoFe protein was representative of the other alpha -96-subst ituted MoFe proteins examined. The incubation of acetylene with the alpha - 96(Leu) MoFe protein decreased the intensity of the normal FeMo-cofactor si gnal 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-dep endent EPR signals observed for the alpha -96(Leu)-substituted MoFe protein were found to follow Curie law I IT dependence, consistent with a ground-s tate transition as observed for FeMo-cofactor. The microwave power dependen ce 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 C-13-labeled cyanide displays a C- 13 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 densit y on the cyanide, and thus suggests that the new component of the cyanide-d ependent EPR signals arise from the direct bonding of cyanide to the FeMo-c ofactor. These data indicate that both acetylene and cyanide are able to in teract with FeMo-cofactor contained within the alpha -96-substituted MoFe p roteins in the resting state. These results support a model where effective interaction of substrates or inhibitors with FeMo-cofactor occurs as a con sequence of both increased reactivity and accessibility of FeMo-cofactor un der turnover conditions. We suggest that, for the wild-type MoFe protein, t he alpha -96(Arg) side chain acts as a gatekeeper, moving during turnover i n order to permit accessibility of acetylene or cyanide to a specific [4Fe- 4S] face of FeMo-cofactor.