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Azotobacter vinelandii Nitrogenases containing altered MoFe proteins with substitutions in the FeMo-Cofactor environment: Effects on the catalyzed reduction of acetylene and ethylene

Fisher, K., Dilworth, M.J., Kim, C-H and Newton, W.E. (2000) Azotobacter vinelandii Nitrogenases containing altered MoFe proteins with substitutions in the FeMo-Cofactor environment: Effects on the catalyzed reduction of acetylene and ethylene. Biochemistry, 39 (11). pp. 2970-2979.

Link to Published Version: http://dx.doi.org/10.1021/bi992092e
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Abstract

Altered MoFe proteins of Azotobacter vinelandii Mo-nitrogenase, with amino acid substitutions in the FeMo-cofactor environment, were used to probe interactions among C2H2, C2H4, CO, and H2. The altered MoFe proteins used were the α-195Asn or α-195Gln MoFe proteins, which have either asparagine or glutamine substituting for α-histidine-195, and the α-191Lys MoFe protein, which has lysine substituting for α-glutamine-191. On the basis of Km determinations, C2H2 was a particularly poor substrate for the nitrogenase containing the α-191Lys MoFe protein. Using C2D2, a correlation was shown between the stereospecificity of proton addition to give the products, cis- and trans-C2D2H2, and the propensity of nitrogenase to produce ethane. The most extensive loss of stereospecificity occurred with nitrogenases containing either the α-195Asn or the α-191Lys MoFe proteins, which also exhibited the highest rate of ethane production from C2H2. These data are consistent with the presence of a common ethylenic intermediate on the enzyme, which is responsible for both ethane production and loss of proton-addition stereochemistry. C2H4 was not a substrate of the nitrogenase with the α-191Lys MoFe protein and was a poor substrate of the nitrogenases incorporating either the wild-type or the α-195Gln MoFe protein, both of which had a low Vmax and high Km (120 kPa). Ethylene was a somewhat better substrate for the nitrogenase with the α-195Asn MoFe protein, which exhibited a Km of 48 kPa and a specific activity for C2H6 formation from C2H4 10-fold higher than the others. Neither the wild-type nitrogenase nor the nitrogenase containing the α-195Asn MoFe protein produced cis-C2D2H2 when turned over under trans-C2D2H2. These results suggest that the C2H4-reduction site is affected by substitution at residue α-195, although whether the effect is related to the substrate-reduction site directly or is mediated through disturbance of the delivery of electrons/protons is unclear. Ethylene inhibited total electron flux, without uncoupling MgATP hydrolysis from electron transfer, to a similar extent for all four A. vinelandii nitrogenases. This observation indicates that this C2H4 flux-inhibition site is remote from the C2H4-reduction site. Added CO eliminated C2H4 reduction but did not fully relieve its electron-flux inhibition with all four A. vinelandii nitrogenases, supporting the suggestion that electron-flux inhibition by C2H4 is not directly connected to C2H4 reduction. Thus, C2H4 has two binding sites, and the presence of CO affects only the site at which it binds as a substrate. When C2H2 was added, it also eliminated C2H6 production from C2H4 and also did not relieve electron-flux inhibition fully. Thus, C2H2 and C2H4 are likely reduced at the same site on the MoFe protein. Two schemes are presented to integrate the results of the interactions of C2H2 and C2H4 with the MoFe proteins.

Publication Type: Journal Article
Publisher: ACS Publications
Copyright: © 2000 American Chemical Society
URI: http://researchrepository.murdoch.edu.au/id/eprint/18363
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