Carbon monoxide binding to α-R277H Mo-nitrogenase - Evidence for multiple pH-dependent species from IR-monitored photolysis

The nitrogenase (N2ase) enzyme family is responsible for the conversion of dinitrogen into biologically accessible ammonia, a critical step in the global nitrogen cycle. Carbon monoxide (CO) has long been known as an inhibitor of dinitrogen reduction, but it can also be reduced to hydrocarbons catalyzed by all three N2ases, namely the wild-type Mo enzyme and select variants and the V and Fe nitrogenases, both of which are orders of magnitude more effective. CO interactions with N2ases are thus relevant to both dinitrogen fixation and FischerTropsch-like chemistry. Here, we investigated the interaction of CO with the alpha-R277H variant of the Azotobacter vinelandii N2ase MoFe protein, in which the alpha-subunit 277Arg residue is replaced by His and results in production of only the S = 3/2 EPR signal (denoted as hi(5)-CO). Fourier-transform infrared (FT-IR) spectroscopy was used to follow the photolysis of CO bound to the alpha-R277H variant under cryogenic conditions. Multiple EPR-silent species were observed with FT-IR spectroscopic signatures previously assigned to CO-inhibited forms of the alpha-H195Q and alpha-H195N N2ase variants. The distribution of these CO-inhibited forms varied dramatically with pH over the range of pH 6.5 to pH 8.5, indicating protonation/deprotonation involvement.

Automated summary

This study investigated the interaction of CO with the alpha-R277H variant of the Azotobacter vinelandii N2ase MoFe protein. It was found that multiple CO-inhibited forms were formed with the variant at different pH values, indicating the involvement of protonation/deprotonation.