We report CW and pulsed Q-band 1,2H ENDOR measurements of intermediate X formed during the assembly of the diferric tyrosyl radical cofactor of the R2 subunit in ribonucleotide reductase. These studies, performed with H2O and D2O buffers, were designed to determine whether the exchangeable proton signals are associated with an hydroxo bridge, a terminal water, or both. In doing so, we identify the types of protonated oxygen (OH x ) species coordinated to the iron ions of X and their disposition relative to the ferric and ferryl iron ions. The exchangeable proton signals displayed by intermediate X belong to two protons associated with a terminal water bound to Fe,III and not to an hydroxo bridge; within the precision of the modeling, this picture of a terminal water is indistinguishable from that of a 2-fold disordered terminal hydroxyl. The fact that X displays strong spin-coupling between iron ions requires that there be one or more oxo/hydroxo bridges. These findings then establish that X contains the [(H x O)FeIIIOFeIV] fragment.

The resting state of nitrogenase shows an S = 3/2 electron paramagnetic resonance (EPR) signal resulting from the FeMo-cofactor (MoFe7S9:homocitrate) of the MoFe protein. When the enzyme undergoes turnover under a CO atmosphere, this signal disappears and two new ones appear:  one under low pressure of CO (denoted lo-CO; 0.08 atm) and the other under high pressure of CO (denoted hi-CO; 0.5 atm). Our recent Q-band (35 GHz) 13C and 57Fe electron nuclear double resonance (ENDOR) studies demonstrated that one CO is bound to the FeMo-cofactor of lo-CO and two to the cofactor of hi-CO. [Christie, P. D.; Lee, H. I.; Cameron, L. M.; Hales, B. J.; Orme-Johnson, W. H.; Hoffman, B. M. J. Am. Chem. Soc. 1996, 118, 8707−8709. Pollack, R. C.; Lee, H. I.; Cameron, L. M.; DeRose, V. J.; Hales, B. J.; Orme-Johnson, W. H.; Hoffman, B. M. J. Am. Chem. Soc. 1995, 117, 8686−8687.] In the present report, we examine the CO-bound FeMo-cofactor in both the lo- and hi-CO forms of the MoFe protein from Azotobacter vinelandii by complete orientation-selective 13C and 1H ENDOR measurements. 1H ENDOR studies reveal that well-resolved signals from a solvent-exchangeable proton seen in the resting state FeMo-cofactor are lost in both of the CO-inhibited forms, indicating a loss in hydrogen bonding as compared to the resting state. This supports the hypothesis that CO binds near the “waist” of the cofactor. Determination of 13C hyperfine tensors of bound 13CO to lo-CO and hi-CO leads to the suggestion that the single CO bound to the FeMo-cofactor of lo-CO may bridge or semibridge two iron ions, while each of the two CO bound to hi-CO is a terminal ligand. These ENDOR measurements and recent FTIR results of Thorneley and co-workers [George, S. J.; Ashby, G. A.; Wharton, C. W.; Thorneley, R. N. F. J. Am. Chem. Soc. 1997, 119, 6450−6451] provide strong mutual support.

Nitrogenase is the metalloenzyme that catalyzes the nucleotide-dependent reduction of N(2), as well as reduction of a variety of other triply bonded substrates, including the alkyne, acetylene. Substitution of the alpha-70(Val) residue in the nitrogenase MoFe protein by alanine expands the range of substrates to include short-chain alkynes not reduced by the unaltered protein. Rapid freezing of the alpha-70(Ala) nitrogenase MoFe protein during reduction of the alkyne propargyl alcohol (HC triple bond CH(2)OH; PA) traps an S = (1)/(2) intermediate state of the active-site metal cluster, the FeMo-cofactor. We have combined CW and pulsed (13)C ENDOR (electron-nuclear double resonance) with two quantitative 35 GHz (1,2)H ENDOR techniques, Mims pulsed ENDOR and the newly devised "stochastic field-modulated" ENDOR, to study this intermediate prepared with isotopically substituted ((13)C, (1,2)H) propargyl alcohol in H(2)O and D(2)O buffers. These measurements allow the first description of a trapped nitrogenase reduction intermediate. The S = (1)/(2) turnover intermediate generated during the reduction of PA contains the 3-carbon chain of PA and exhibits resolved (1,2)H ENDOR signals from three protons, two strongly coupled (H(a)) and one weakly coupled (H(b)); H(a)(c) originates as the C3 proton of PA, while H(a)(s) and H(b) are solvent-derived. The two H(a) protons have identical hyperfine tensors, despite having different origins. The equality of the (H(a)(s), H(a)(c)) hyperfine tensors strongly constrains proposals for the structure of the cluster-bound reduced PA. Through consideration of model structures found in the Cambridge Structural Database, we propose that the intermediate contains a novel bio-organometallic complex in which a reduction product of propargyl alcohol binds as a metalla-cyclopropane ring to a single Fe atom of the Fe-S face of the FeMo-cofactor that is composed of Fe atoms 2, 3, 6, and 7. Of the two most attractive structures, one singly reduced at C3 (4), the other being the doubly reduced allyl alcohol product (6), we tentatively favor 6 because of the "natural" assignment it affords for H(b).