Formyltetrahydrofolate Decarbonylase Synthesizes the Active Site CO Ligand of O₂-Tolerant [NiFe] Hydrogenase

Abstract

[NiFe] hydrogenases catalyze the reversible oxidation of molecular hydrogen into two protons and two electrons. A key organometallic chemistry feature of the NiFe active site is that the iron atom is co-coordinated by two cyanides (CN–) and one carbon monoxide (CO) ligand. Biosynthesis of the NiFe­(CN)2(CO) cofactor requires the activity of at least six maturation proteins, designated HypA–F. An additional maturase, HypX, is required for CO ligand synthesis under aerobic conditions, and preliminary in vivo data indicated that HypX releases CO using N 10-formyltetrahydrofolate (N 10-formyl-THF) as the substrate. HypX has a bipartite structure composed of an N-terminal module similar to N 10-formyl-THF transferases and a C-terminal module homologous to enoyl-CoA hydratases/isomerases. This composition suggested that CO production takes place in two consecutive reactions. Here, we present in vitro evidence that purified HypX first transfers the formyl group of N 10-formyl-THF to produce formyl-coenzyme A (formyl-CoA) as a central reaction intermediate. In a second step, formyl-CoA is decarbonylated, resulting in free CoA and carbon monoxide. Purified HypX proved to be metal-free, which makes it a unique catalyst among the group of CO-releasing enzymes.

A membrane‐bound [NiFe]‐hydrogenase large subunit precursor whose C‐terminal extension is not essential for cofactor incorporation but guarantees optimal maturation

“…First, the Fe(CN) 2 (CO) moiety is assembled with the aid of the HypE and HypF proteins, which synthesize the cyanide ligands out of carbamoyl phosphate (Blokesch et al, 2004; Reissmann et al, 2003). The metabolic origin of CO under anaerobic conditions remains, however, unclear (Bürstel et al, 2011; Nutschan, Golbik, & Sawers, 2019), while under aerobic conditions, this diatomic ligand is derived from formyltetrahydrofolate (Bürstel et al, 2016; Schulz et al, 2020). Assembly takes place on a scaffold complex, consisting of the HypC and HypD proteins, from which the Fe(CN) 2 (CO) moiety is transferred to the apo‐large subunit (Bürstel et al, 2012; Stripp et al, 2013).…”

Electron inventory of the iron-sulfur scaffold complex HypCD essential in [NiFe]-hydrogenase cofactor assembly

“…It has been demonstrated that the HypCD complex catalyzes the hydrolysis of ATP [ 21 ], which would help to overcome such thermodynamic barriers, not unlike CO dehydrogenase [ 59 ], the ‘iron protein’ of nitrogenase [ 60 ], or enoyl-CoA reductase [ 61 ]. Under aerobic conditions, the CO ligand is generated by decarbonylating formyltetrahydrofolate [ 62 , 63 ]. However, the metabolic source in strict or facultative anaerobes must be different.…”

Electron Inventory of the Iron-Sulfur Scaffold Complex HypCD Essential in [NiFe]-Hydrogenase Cofactor Assembly

“…31 It has been demonstrated that the HypCD complex catalyzes the hydrolysis of ATP 21 * nitrogenase, or enoyl-CoA reductase. [58][59][60] Under aerobic conditions, the CO ligand is generated by decarbonylating formyltetrahydrofolate 22,23 , but the metabolic source in strict or facultative anaerobes must be different. To this end, our spectroscopic evaluation of the HypCD scaffold complex provides comprehensive evidence for an electron inventory fit to drive multi-electron redox reactions.…”

“…Depending on the host organism, the CO ligand may stem from different metabolic sources; e.g., HypX from Cupriavidus necator has been shown to yield CO by decarbonylating formyltetrahydrofolate under aerobic conditions. 22,23 The CNligands are generated by the maturation proteins HypE and HypF and attached to the CO-modified iron ion, forming the [Fe](CN)2CO cofactor precursor on the HypCD complex ( Figure 1B). 24,25 A HypD HypC Figure 2A shows an overlay of the crystalized maturation protein HypD from Thermococcus kodakaraensis (TkHypD) with a computational model of HypC from Rhizobium leguminosarum (RlHypC) carrying the [Fe](CN)2CO cofactor precursor.…”