Understanding substrate and inhibitor interactions with Mo-nitrogenase

The nitrogenase enzyme is a metalloenzyme that plays a key role in the biological nitrogen cycle. Nitrogenase fixes nitrogen by catalysing the reduction of dinitrogen in the atmosphere to ammonia. The enzyme is a protein expressed by free-living soil bacteria and symbiotic bacteria living in the roots of certain plants. The dinitrogen reduction mechanism of the enzyme remains poorly understood. This thesis describes using electrochemistry and Infrared spectroscopy to study the mechanism of molybdenum (Mo)-nitrogenase. In order to overcome the complexity of electron transfer by the native reductant Fe protein coupled to the hydrolysis of ATP, a carbon working electrode was used instead of Fe protein as a reductant to supply electrons to MoFe protein. A series of known inhibitors and different possible substrates of nitrogenase (including protons, dinitrogen, acetylene, isocyanides and carbon monoxide) was introduced into this spectroelectrochemical study. Meanwhile, genetic variants of nitrogenase were also studied to determine the role of specific amino acids in the protein by this approach.
Following the previous study of nitrogenase MoFe protein in the Vincent group, an attachment approach was developed to immobilise protein onto the carbon working electrode in the spectroelectrochemical cell. Consistent with an earlier electrochemical study of MoFe protein trapped in Nafion, the attached MoFe protein was shown to commence proton reduction at about -0.9 V. The interaction between MoFe protein and inhibitor, carbon monoxide was studied by IR spectroelectrochemistry. The results from both infrared spectroscopy and electrochemistry were consistent with a previous study under catalytic turnover conditions, and also showed that CO binds to the MoFe protein at around -0.9 V and below. For the first time direct electron transfer from a carbon working electrode to one of the variants of the nitrogenase MoFe protein (α-64Tyr→His) was observed, together with preliminary evidence of electron transfer to the wild type MoFe protein.
Acetylene is known to be a non-natural substrate of nitrogenase, and was shown to inhibit the proton reduction current by the MoFe protein at -0.9 V and below. Inhibition was irreversible when CO was flushed out with Ar at -0.9 V, but Ar was able to recover some of the proton reduction activity at more negative potentials. The product ethylene was followed by GC analysis, which confirmed acetylene reduction by MoFe protein at about -0.9 V and more negative.
When isocyanide was introduced to MoFe protein, a shift in the peak of v(NC) from isocyanide to higher wavenumber compared to that in free solution was observed even at a mild potential. This suggests that isocyanide binds to a more oxidized redox state of the MoFe protein compared to the state needed for proton reduction, CO binding and acetylene reduction. Preliminary evidence for methylamine as the product was obtained by GC at much lower potentials.
These results establish, for the first time, the potentials at which different ligands bind to the MoFe protein, and thus should help in establishing aspects of the mechanism of substrate reduction by nitrogenase.

Keywords: nitrogenase
spectroelectrochemistry
MoFe protein
Advisor: Vincent, K