Role of the MoFe Protein ß-95-Cysteinyl Residue in Nitrogenase Catalysis in Azotobacter vinelandii

Previous studies revealed that ß-95-Cys provides an
essential ligand to one of the Fe atoms on the P cluster
within the MoFe protein of nitrogenase, and a limited number
of substitutions at this position resulted in
inactive nitrogenase. It was also found that the
counterpart of ß-95-Cys, Alpha-88-Cys, which also
acts as
a cysteinyl ligand to the P cluster, is replaceable
without a complete loss of activity. In order to study
the structure-function relationship of the
protein environment in this region with respect to
the P-cluster, subtle changes were introduced at
ß-95-Cys
in Azotobacter vinelandiinitrogenase through
site-directed
mutagenesis and gene replacement method. Some
crude extracts from the mutants with substitutions at
ß-95-Cys contain typical FeMo cofactor EPR signal.
The ß-95^Asp MoFe protein also has
significant nitrogenase
activity, but lower, suggesting that ß-95-Cys is not
absolutely required for both FeMo cofactor insertion
and nitrogenase activity.

In order to characterize its catalytic features, the
ß-95^Asp MoFe protein was purified from mutant strain
DJ1096.
It has significantly reduced H⁺ reduction,
C₂H₂-reduction
and N₂-reduction activity.
It was found that a higher
percentage of electron flux goes to H⁺
compared to the wild
type MoFe protein. It was also found that reductant
independent ATP hydrolysis occurs during H⁺
reduction,
suggesting that the altered MoFe protein has an increased
affinity for Fe protein-ADP complex. Surprisingly,
CO has a significant enhancement effect on H⁺
reduction at
low electron flux, but not at high electron flux, and
highly couples the electron transfer to ATP hydrolysis.
These results indicate that the binding of CO to the MoFe
protein may either decrease the affinity of Fe-ADP complex
for the ß-95^Asp MoFe protein or
facilitate electron
acceptance by the P cluster, thus improving the electron
transfer to substrate.