ATP-uncoupled, six-electron photoreduction of hydrogen cyanide to methane by the molybdenum-iron protein.

A detailed study of the eight-electron/eight-proton catalytic reaction of nitrogenase has been hampered by the fact that electron and proton flow in this system is controlled by ATP-dependent protein-protein interactions. Recent studies have shown that it is possible to circumvent the dependence on ATP through the use of potent small-molecule reductants or light-driven electron injection, but success has been limited to two-electron reductions of hydrazine, acetylene, or protons. Here we show that a variant of the molybdenum-iron protein labeled with a Ru-photosensitizer can support the light-driven, six-electron catalytic reduction of hydrogen cyanide into methane and likely also ammonia. Our findings suggest that the efficiency of this light-driven system is limited by the initial one- or two-electron reduction of the catalytic cofactor (FeMoco) to enable substrate binding, but the subsequent electron-transfer steps into the FeMoco-bound substrate proceed efficiently.

X-ray crystallography.

X-ray crystallography has been particularly important in the study of the enzyme nitrogenase, providing researchers with high-resolution structural models that have been essential to studying the enzyme's unique metal clusters and nucleotide-binding modes and protein interactions. While several important nitrogenase structures have already been determined using X-ray crystallography, the technique still holds great potential for future significant discoveries involving reaction intermediates and redox states of the enzyme's metal clusters. Thus, it is important to inform future nitrogenase researchers about the procedures for obtaining crystals of nitrogenase component proteins and their complexes and determining their structures. While nitrogenase component proteins from several bacteria have been crystallized, the majority of structures and those of highest resolution are of the nitrogenase proteins from the bacteria Azotobacter vinelandii. Therefore, the bulk of this chapter will focus on methods for crystallization and structure determination of nitrogenase component proteins from A. vinelandii.

ATP- and iron-protein-independent activation of nitrogenase catalysis by light.

We report here the light-driven activation of the molybdenum-iron-protein (MoFeP) of nitrogenase for substrate reduction independent of ATP hydrolysis and the iron-protein (FeP), which have been believed to be essential for catalytic turnover. A MoFeP variant labeled on its surface with a Ru-photosensitizer is shown to photocatalytically reduce protons and acetylene, most likely at its active site, FeMoco. The uncoupling of nitrogenase catalysis from ATP hydrolysis should enable the study of redox dynamics within MoFeP and the population of discrete reaction intermediates for structural investigations.