[PubMed] [Google Scholar] 89. from recent success in trapping substrates and inhibitors in the active site metallic cluster FeMo-cofactor, and finally, considerations of the mechanism of N2 reduction catalyzed by nitrogenase. that is resistant to acetylene inhibition of N2 fixation-dependent growth (78). Considering that acetylene is only slightly larger than N2 (Number 5), it would have been impossible to rationally design the appropriate residue position for substitution. Thus, the beauty of the genetic approach was that the organism did all the hard work by simple genetic selection. A number of spontaneous acetylene-resistant mutants were isolated and characterized, and all of them experienced substitutions in the -69Gly position (78). For these substituted MoFe proteins, the and for N2 reduction were unchanged, whereas the for acetylene reduction experienced increased 20-collapse AZD5423 (79). The -69Gly residue is located in the second shell of amino acids away from those directly interacting with the FeS face in the waist of FeMo-cofactor(Number 6). An adjacent amino acid in the -chain is definitely -70Val, the side-chain of which approaches one of the three 4Fe-4S faces of FeMo-cofactor. This FeS face of FeMo-cofactor entails Fe atoms 2, 3, 6, and 7 (using the numbering plan in the PDB file 1M1N). Because -69Gly is not close enough to directly influence substrate binding, it is more reasonable to expect that substitutions placed in the -69Gly position might alter the dynamic movement of the -70Val side-chain therefore permitting the discrimination between acetylene and N2 or effective access to this 4Fe face. This work offered the first direct experimental evidence that initial substrate binding for both acetylene and N2 can occur at a specific Fe-S face of FeMo-cofactor. Altering the substrate size range The FeS face of FeMo-cofactor hypothesized to be the site of reduction is directly approached by the side chain of -70Val. This suggested a model wherein the side chain of this residue might impose steric constraints AZD5423 on the size of substrates that could gain access to the active site. To test this hypothesis, the -70Val was substituted by amino acids having either larger (Ile) or smaller (Ala or Gly) part chains (Number 7) and the ability of the substituted MoFe proteins to reduce substrates of different sizes was tested (80C84). It was expected that substitution of the larger part chain of -70Ile might block access of substrates, whereas substitution by the smaller part chain -70Ala might open access to the active site and therefore permit larger compounds that are not normally substrates to become substrates for nitrogenase. A series of kinetic studies supported this model (80C85). The -70Ala MoFe protein was found to reduce propyne and propargyl alcohol (Number 5) at substantial rates, whereas the -70Val (wild-type) MoFe protein did not. It was also found that the -70Ala substituted MoFe protein can reduce hydrazine (H2N-NH2) at high rates, whereas the wild-type does not (86). Substitution of -70Val by the smaller Gly residue also endowed the MoFe protein with an ability to reduce an even larger alkyne substrate, 1-butyne, at measureable rates (84). Inside a complementary experiment, the -70Ile substituted MoFe protein was found to reduce only protons at normal rates (validating the catalytic integrity of the active site), with limited reduction rates for acetylene, N2, or hydrazine (87). These findings reveal two essential elements about substrate relationships with AZD5423 nitrogenase: (i) both alkyne and nitrogenous substrates, including acetylene and N2, have the capacity to interact with the same FeS face of FeMo-cofactor that is approached from the -70Val part chain and (ii) the substrate range of nitrogenase can be controlled by manipulation of the side chain located in the -70 residue position. Open in a separate window Number 7 Control of substrate access to FeMo-cofactor. Shown is the FeMo-cofactor without R-homocitrate viewed down the Mo end. The side chain of -70Val is definitely shown having a Vehicle der Waals mesh (remaining). Also demonstrated are computer generated models of the Vehicle der Waals surface for -70Ala (center) and -70Ile (ideal) substituted MoFe proteins. While the studies explained above narrowed the location of connection for both alkyne and nitrogenous substrates to a specific FeS face of FeMo-cofactor, they did not predict a specific site for binding. Additional studies helped to further localize the binding location for alkyne substrates. AZD5423 One study took advantage of the observation that propargyl alcohol is a substrate for the -70Ala substituted MoFe protein and that AZD5423 during turnover with KIAA0700 this substrate, a novel EPR active state could be caught by rapidly freeze-quenching (81, 83). As explained below, through use of electron nuclear double resonance (ENDOR) spectroscopy, it was possible to demonstrate that this EPR spectrum results from FeMo-cofactor that has a reduction product of propargyl alcohol bound.