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J Biol Chem, Vol. 275, Issue 15, 11459-11464, April 14, 2000

Isolation and Characterization of an Acetylene-resistant Nitrogenase^*

Jason Christiansen, Valerie L. Cash, Lance C. Seefeldt^§¶, and Dennis R. Dean^¶

From the  Department of Biochemistry, Virginia Tech, Blacksburg, Virginia 24061-0346 and ^§ Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322

A genetic strategy was developed for the isolation of a mutant strain of Azotobacter vinelandii that exhibits in vivo nitrogenase activity resistant to inhibition by acetylene. Examination of the kinetic features of the altered nitrogenase MoFe protein produced by this strain, which has serine substituted for the -subunit Gly⁶⁹ residue, is consistent with other studies that indicate the MoFe protein normally contains at least two acetylene binding/reduction sites. The first of these is a high affinity site and is the one primarily accessed during typical acetylene reduction assays. Results of the present work indicate that this acetylene binding/reduction site is not directly relevant to the mechanism of nitrogen reduction because it can be eliminated or severely altered without significantly affecting nitrogen reduction. Elimination of this site also results in the manifestation of a low affinity acetylene-binding site to which both acetylene and nitrogen are able to bind with approximately the same affinity. In contrast to the normal enzyme, nitrogen and acetylene binding to the altered MoFe protein are mutually competitive. The location of the -Ser⁶⁹ substitution is interpreted to indicate that the 4Fe-4S face of the FeMo cofactor capped by the -subunit Val⁷⁰ residue is the most likely region within FeMo cofactor to which acetylene binds with high affinity.

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