Isolation and characterization of an acetylene-resistant nitrogenase

A genetic strategy was developed for the isolation of a mutant strain of Az otobacter vinelandii that exhibits in vivo nitrogenase activity resistant t o inhibition by acetylene, Examination of the kinetic features of the alter ed nitrogenase MoFe protein produced by this strain, which has serine subst ituted for the alpha-subunit Gly(69) residue, is consistent with other stud ies 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. Resu lts 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 nitr ogen reduction. Elimination of this site also results in the manifestation of a low affinity acetylene-binding site to which both acetylene and nitrog en are able to bind with approximately the same affinity. In contrast to th e normal enzyme, nitrogen and acetylene binding to the altered MoFe protein are mutually competitive. The location of the alpha-Ser(69) substitution i s interpreted to indicate that the 4Fe-4S face of the FeMo cofactor capped by the cu-subunit Val(70) residue is the most likely region within FeMo cof actor to which acetylene binds with high affinity.