Assembly of the H-cluster of [FeFe]-hydrogenase

Hydrogenases are enzymes that very efficiently interconvert protons/electrons and hydrogen gas (H2), and thus are of great interest for their potential applications for bioenergy production. These enzymes employ special organometallic cofactors that perform the H2 oxidation/formation chemistry, featuring cheap, high-abundance metals rather than expensive, rare metals such as platinum. Indeed, much research effort is focused on developing suitable replacements for platinum-dependent catalysis, and hydrogenase enzymes provide important paradigms for electrocatalytic formation of H2 for renewable energy applications. A major aim here is the coupling of photosynthetic (light-activated source of electrons) and hydrogenase systems to produce H2 directly from water (from which electrons and protons are sourced).
[FeFe]-hydrogenase is the most efficient hydrogenase for H2 production, generating thousands of molecules per second. Its active site cofactor is called the ‘H-cluster’, consisting of a ‘regular’ [4Fe-4S] cluster bridged to a unique dinuclear iron site, called [2Fe]H, in which each of the irons is coordinated to carbon monoxide (CO) and cyanide (CN-) ligands, and are bridged by a third CO and a sulfur/nitrogen linker. A major feature that has emerged over the past 30 years is that complex metal-containing cofactors require complex cellular assembly machineries. This is certainly the case for the H-cluster, particularly because it requires the synthesis of CO and CN-, which are well-known cellular toxins.
While the [4Fe-4S] cluster is synthesised by the general housekeeping iron-sulfur cluster assembly machinery, the [2Fe]H subcluster requires a dedicated machinery consisting of at least three enzymes, HydG, HydE and HydF, that work together to sequentially construct the subcluster, starting from iron, cysteine (the source of the bridging sulfur), and tyrosine (the source of CO and CN-). While a lot is now known about how the subcluster is synthesised, many important questions remain, concerning precisely what reactions the assembly enzymes catalyse and what intermediates in the assembly process they pass on to the next partner in the assembly line.
In this application, we propose to apply a novel methodology to answer some of these key questions. Mass spectrometry (MS) is one of the most widespread analytical methods in biology/biochemistry. A particular type of MS, called native MS, facilitates ionisation of proteins in a folded state such that any non-covalently attached cofactors are retained by the protein and are consequently measured. Thus, the additional mass due to the cofactor(s) is detected and, in combination with more traditional spectroscopic and structural approaches, unambiguous identification of bound intermediates and products can be achieved. We have previously developed native MS for studies of iron-sulfur cluster proteins that sense important environmental cues such as presence of O2 or various stresses.  In preliminary experiments we have obtained data for all three assembly enzymes in which cofactors and reaction intermediates/products are detected, including following transfer from one enzyme to another, demonstrating the feasibility of our approach. By initiating reactions via addition of substrates and other necessary reaction components, and the use of isotope substitutions that result in mass shifts that depend on elemental constituents, we will identify reaction intermediates and products and follow their formation/decay in real time. Mixing of assembly proteins will enable characterisation of molecules that are transferred from one to the other. By placing these observations into the context of already published results, our data will provide a step-change in understanding of [FeFe]-hydrogenase assembly.