De novo modeling of the F(420)-reducing [NiFe]-hydrogenase from a methanogenic archaeon by cryo-electron microscopy.

Deryck J Mills,Seigo Shima,Mike Strauss,Janet Vonck,Stella Vitt

doi:10.7554/elife.00218

Abstract

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. DOI:http://dx.doi.org/10.7554/eLife.00218.001.

Highlights

F420-reducing [NiFe]-hydrogenase (Frh) accounts for about 1% of the cytoplasmic protein and has been shown by electron microscopy to form a huge complex of similar appearance in all species investigated: Methanococcus voltae (Muth et al, 1987), Methanospirillum hungatei (Sprott et al, 1987), Methanobacterium thermoautotrophicum ΔH (Wackett et al, 1987), and Methanobacterium thermoautotrophicum Marburg (Braks et al, 1994)
The Frh complex was highly purified from M. marburgensis under strict anaerobic conditions in the presence of FAD
We found that all projections of the Frh complex are ring-shaped with a diameter of ∼16 nm (Figure 1A)

Summary

Introduction

[NiFe]-hydrogenases are microbial enzymes that heterolytically cleave H2, after which the electrons from a hydride ion are reversibly transferred to electron carriers These enzymes are involved in many metabolic pathways in microbial ecosystems, notably in methanogenesis. 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. Developments in instrumentation and image processing software have made it possible to determine structures of large macromolecular complexes to near-atomic resolution by cryo-electron microscopy. The location of all cofactors were identified, and the backbone of the three proteins was traced, including FrhB that has a novel fold

Results

Discussion

Materials and Methods

Funding Funder Max Planck Society