Methanogenic archaea use a [NiFe]-hydrogenase, Frh, for oxidation/reduction of F420, an important hydride carrier in the methanogenesis pathway from H2 and CO2. Frh accounts for about 1% of the cytoplasmic protein and forms a huge complex consisting of FrhABG heterotrimers with each a [NiFe] center, four Fe-S clusters and an FAD. Here, we report the structure determined by near-atomic resolution cryo-EM of Frh with and without bound substrate F420. The polypeptide chains of FrhB, for which there was no homolog, was traced de novo from the EM map. The 1.2-MDa complex contains 12 copies of the heterotrimer, which unexpectedly form a spherical protein shell with a hollow core. The cryo-EM map reveals strong electron density of the chains of metal clusters running parallel to the protein shell, and the F420-binding site is located at the end of the chain near the outside of the spherical structure.
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.