Decision letter: De novo modeling of the F420-reducing [NiFe]-hydrogenase from a methanogenic archaeon by cryo-electron microscopy

Abstract

Many microbes grow by producing methane gas from carbon dioxide and hydrogen gas, and enzymes known as hydrogenases play important roles in this metabolic process. The production of methane in these microbes depends on a nickel–iron hydrogenase called Frh adding electrons to a coenzyme called F420. This hydrogenase cleaves a hydrogen molecule into two electrons, which are transferred to the F420 coenzyme, and two protons. The reduced form of F420 is then used for several reactions in the methane production process. This process, which is known as methanogenesis, provides the microbes with energy. Nickel–iron hydrogenases can be divided into five different groups, but researchers have been able to determine the detailed structures of the enzymes in just one of these groups. All nickel–iron hydrogenases contain at least two subunits: a large subunit with a catalytic center composed of both nickel and iron ions and a small subunit that contains three iron–sulfur clusters. Frh—which is short for F420-reducing nickel–iron hydrogenase—is known to have a third subunit comprising an extra iron–sulfur cluster and a coenzyme called FAD that allows it to interact with the F420 coenzyme. However, until now, little was known about the detailed structure of the Frh enzyme. Mills et al. have used electron cryo-microscopy (cryo-EM) to determine the structure of Frh when it is on its own, and also when it is bound to F420. This technique involves freezing a solution of the enzyme in a thin layer of ice and recording an image of this layer in an electron microscope. By combining a large number of images, each of which contains many identical enzymes in different orientations, it is possible to determine the 3-dimensional structure of the enzyme. Mills et al. found that Frh forms a very large tetrahedral complex that contains six Frh dimers. And by comparing the structure with and without F420, they identify a pocket near the FAD coenzyme that the F420 coenzyme binds to. They also identify a fold in the third subunit that allows proteins to bind both FAD and F420. The work demonstrates the potential of cryo-EM to elucidate structures that cannot be determined by other approaches.