Impact of the second coordination sphere of the molybdopterin cofactor on the mechanism of formate dehydrogenase from <i>Cupriavidus necator</i>

Molybdenum containing enzymes play a pivotal role in the global carbon and nitrogen cycles using a common molybdopterin cofactor. Mechanistic studies have revealed a great deal about molybdenum enzymes but have yet to detail the impact the secondary binding interactions have on catalysis. Herein, we describe a double variant of formate dehydrogenase from <i>Cupriavidus necator</i> (<i>Cn</i>Fds) that changes the electrostatic and hydrogen bonding to the ligands to molybdenum resulting in a complete loss of formate oxidation activity, which occurs by outer sphere hydride transfer, and gain of nitrate reduction activity, which is proposed to follow an inner sphere oxygen atom transfer mechanism. We have assigned these observed changes to the stability of the terminal ligand which in turn directs the catalytic outcome. The results here illustrate the importance of the secondary sphere interactions in directing oxygen atom transfer <i>vs.</i> hydride transfer mechanisms in molybdenum containing enzymes.