Towards artificial methanogenesis: biosynthesis of the [Fe]-hydrogenase cofactor and characterization of the semi-synthetic hydrogenase

Abstract

The greenhouse gas and energy carrier methane is produced on Earth mainly by methanogenic archaea. In the hydrogenotrophic methanogenic pathway the reduction of one CO to one methane molecule requires four molecules of H containing eight electrons. Four of the electrons from two H are supplied for reduction of an electron carrier F, which is catalyzed by F-reducing [NiFe]-hydrogenase under nickel-sufficient conditions. The same reaction is catalysed under nickel-limiting conditions by [Fe]-hydrogenase coupled with a reaction catalyzed by F-dependent methylene tetrahydromethanopterin dehydrogenase. [Fe]-hydrogenase contains an iron-guanylylpyridinol (FeGP) cofactor for H activation at the active site. Fe of FeGP is coordinated to a pyridinol-nitrogen, an acyl-carbon, two CO and a cysteine-thiolate. We report here on comparative genomic analyses of biosynthetic genes of the FeGP cofactor, which are primarily located in a hmd-co-occurring (hcg) gene cluster. One of the gene products is HcgB which transfers the guanosine monophosphate (GMP) moiety from guanosine triphosphate (GTP) to a pyridinol precursor. Crystal structure analysis of HcgB from Methanococcus maripaludis and its complex with 6-carboxymethyl-3,5-dimethyl-4-hydroxy-2-pyridinol confirmed the physiological guanylyltransferase reaction. Furthermore, we tested the properties of semi-synthetic [Fe]-hydrogenases using the [Fe]-hydrogenase apoenzyme from several methanogenic archaea and a mimic of the FeGP cofactor. On the basis of the enzymatic reactions involved in the methanogenic pathway, we came up with an idea how the methanogenic pathway could be simplified to develop an artificial methanogenesis system.

Biomimetic Hydrogenation Catalyzed by a Manganese Model of [Fe]‐Hydrogenase

“…In nature, hydrogenation of methenyl-H 4 MPT + gives methylene-H 4 MPT, which serves as a hydride donor for the reduction of F420 (8-hydroxy-5-deazaflavin) coenzyme to form F420H 2 , an essential electron carrier for hydrogenotrophic methanogenesis. [14] The function of methylene-H 4 MPT is similar to that of NADH and NADPH in biological hydridetransfer reactions. [15] We thought that catalyst 5 might be suitable for the hydrogenation of substrates mimicking methenyl-H 4 MPT + or NAD + /NADP + , yielding hydride donors that could be further utilized for the reduction of another unsaturated molecule.…”

“…Encouraged by the above results, we considered further applications of catalyst 5 in biomimetic hydrogenation. In nature, hydrogenation of methenyl‐H 4 MPT + gives methylene‐H 4 MPT, which serves as a hydride donor for the reduction of F420 (8‐hydroxy‐5‐deazaflavin) coenzyme to form F420H 2 , an essential electron carrier for hydrogenotrophic methanogenesis . The function of methylene‐H 4 MPT is similar to that of NADH and NADPH in biological hydride‐transfer reactions .…”

Heterologous Hydrogenase Overproduction Systems for Biotechnology—An Overview

“…At least seven hmd co-occurring genes ( hcgA-G ) are required for FeGP cofactor synthesis [ 30 , 38 , 39 ]. So far, the functions of five of them, HcgB, HcgC, HcgD, HcgE and HcgF, in FeGP cofactor biosynthesis have been elucidated [ 40 , 41 , 42 , 43 , 44 ]. The functions of HcgA and HcgG are, however, still not known, and only a putative role in CO ligand synthesis or pyridinol assembly is proposed [ 42 ].…”

“…So far, the functions of five of them, HcgB, HcgC, HcgD, HcgE and HcgF, in FeGP cofactor biosynthesis have been elucidated [ 40 , 41 , 42 , 43 , 44 ]. The functions of HcgA and HcgG are, however, still not known, and only a putative role in CO ligand synthesis or pyridinol assembly is proposed [ 42 ]. The seven Hcg proteins are, in contrast to the Hmd-paralogue HmdII, essential for Fe hydrogenase maturation [ 39 ].…”

The Role of Mass Spectrometry in Structural Studies of Flavin-Based Electron Bifurcating Enzymes

“…In addition, Chowdhury et al (2016) have used ICP to demonstrate that Acidaminococcus fermentans ferredoxin/flavodoxin-NAD + reductase (Rnf) activity is dependent on the concentration of Na + ions. Apart from protein identification, MALDI-TOFMS has been used for identification of a variety of reaction product(s) ( Chowdhury et al, 2014 ; Bai et al, 2017 ). Recently, the methods, which are capable of providing information on protein structure, dynamics, and connectivity in its native environment (including in vivo studies) have become a primary choice.…”