HydG, the "dangler" iron, and catalytic production of free CO and CN-: implications for [FeFe]-hydrogenase maturation.

The organometallic H-cluster of the [FeFe]-hydrogenase consists of a [4Fe-4S] cubane bridged via a cysteinyl thiolate to a 2Fe subcluster ([2Fe]H) containing CO, CN-, and dithiomethylamine (DTMA) ligands. The H-cluster is synthesized by three dedicated maturation proteins: the radical SAM enzymes HydE and HydG synthesize the non-protein ligands, while the GTPase HydF serves as a scaffold for assembly of [2Fe]H prior to its delivery to the [FeFe]-hydrogenase containing the [4Fe-4S] cubane. HydG uses l-tyrosine as a substrate, cleaving it to produce p-cresol as well as the CO and CN- ligands to the H-cluster, although there is some question as to whether these are formed as free diatomics or as part of a [Fe(CO)2(CN)] synthon. Here we show that Clostridium acetobutylicum (C.a.) HydG catalyzes formation of multiple equivalents of free CO at rates comparable to those for CN- formation. Free CN- is also formed in excess molar equivalents over protein. A g = 8.9 EPR signal is observed for C.a. HydG reconstituted to load the 5th "dangler" iron of the auxiliary [4Fe-4S][FeCys] cluster and is assigned to this "dangler-loaded" cluster state. Free CO and CN- formation and the degree of activation of [FeFe]-hydrogenase all occur regardless of dangler loading, but are increased 10-35% in the dangler-loaded HydG; this indicates the dangler iron is not essential to this process but may affect relevant catalysis. During HydG turnover in the presence of myoglobin, the g = 8.9 signal remains unchanged, indicating that a [Fe(CO)2(CN)(Cys)] synthon is not formed at the dangler iron. Mutation of the only protein ligand to the dangler iron, H272, to alanine nearly completely abolishes both free CO formation and hydrogenase activation, however results show this is not due solely to the loss of the dangler iron. In experiments with wild type and H272A HydG, and with different degrees of dangler loading, we observe a consistent correlation between free CO/CN- formation and hydrogenase activation. Taken in full, our results point to free CO/CN-, but not an [Fe(CO)2(CN)(Cys)] synthon, as essential species in hydrogenase maturation.

H-cluster assembly intermediates built on HydF by the radical SAM enzymes HydE and HydG.

[FeFe]-hydrogenase catalyzes the reversible reduction of protons to H2 at a complex metallocofactor site, the H-cluster. Biosynthesis of this active-site H-cluster requires three maturation enzymes: the radical S-adenosylmethionine enzymes HydE and HydG synthesize the nonprotein ligands, while the GTPase HydF provides a scaffold for assembly of the 2Fe subcluster of the H-cluster ([2Fe]H) prior to its transfer to hydrogenase. To delineate the assembly and delivery steps for the 2Fe precursor cluster coordinated to HydF ([2Fe]F), we have heterologously expressed HydF in the presence of HydE alone (HydFE) or HydG alone (HydFG), and characterized the resulting purified HydFE and HydFG using UV-visible, EPR, and FTIR spectroscopies and biochemical assays. The iron-sulfur clusters on HydF are modified by co-expression with HydE or HydG, as evidenced by the changes in the visible, EPR, and FTIR spectral features. Further, biochemical assays show that HydFE is capable of activating HydAΔEFG to a limited extent (~ 1% of WT) even though the normal source of CO and CN- ligands of [2Fe]H (HydG) was absent. Activation assays performed with HydFG, in contrast, exhibit no ability to mature HydAΔEFG. It appears that in the case of HydFE, trace diatomics from the cellular environment are incorporated into a [2Fe]F-like precursor on HydF in the absence of HydG. We conclude that the product of HydE, presumably the dithiomethylamine ligand of [2Fe]H, is absolutely essential to the activation process, while the diatomic products of HydG can be provided from alternate sources.

Electron Spin Relaxation and Biochemical Characterization of the Hydrogenase Maturase HydF: Insights into [2Fe-2S] and [4Fe-4S] Cluster Communication and Hydrogenase Activation.

Nature utilizes [FeFe]-hydrogenase enzymes to catalyze the interconversion between H2 and protons and electrons. Catalysis occurs at the H-cluster, a carbon monoxide-, cyanide-, and dithiomethylamine-coordinated 2Fe subcluster bridged via a cysteine to a [4Fe-4S] cluster. Biosynthesis of this unique metallocofactor is accomplished by three maturase enzymes denoted HydE, HydF, and HydG. HydE and HydG belong to the radical S-adenosylmethionine superfamily of enzymes and synthesize the nonprotein ligands of the H-cluster. These enzymes interact with HydF, a GTPase that acts as a scaffold or carrier protein during 2Fe subcluster assembly. Prior characterization of HydF demonstrated the protein exists in both dimeric and tetrameric states and coordinates both [4Fe-4S]2+/+ and [2Fe-2S]2+/+ clusters [Shepard, E. M., Byer, A. S., Betz, J. N., Peters, J. W., and Broderick, J. B. (2016) Biochemistry 55, 3514-3527]. Herein, electron paramagnetic resonance (EPR) is utilized to characterize the [2Fe-2S]+ and [4Fe-4S]+ clusters bound to HydF. Examination of spin relaxation times using pulsed EPR in HydF samples exhibiting both [4Fe-4S]+ and [2Fe-2S]+ cluster EPR signals supports a model in which the two cluster types either are bound to widely separated sites on HydF or are not simultaneously bound to a single HydF species. Gel filtration chromatographic analyses of HydF spectroscopic samples strongly suggest the [2Fe-2S]+ and [4Fe-4S]+ clusters are coordinated to the dimeric form of the protein. Lastly, we examined the 2Fe subcluster-loaded form of HydF and showed the dimeric state is responsible for [FeFe]-hydrogenase activation. Together, the results indicate a specific role for the HydF dimer in the H-cluster biosynthesis pathway.

A Redox Active [2Fe-2S] Cluster on the Hydrogenase Maturase HydF.

[FeFe]-hydrogenases are nature's most prolific hydrogen catalysts, excelling at facilely interconverting H2 and protons. The catalytic core common to all [FeFe]-hydrogenases is a complex metallocofactor, referred to as the H-cluster, which is composed of a standard [4Fe-4S] cluster linked through a bridging thiolate to a 2Fe subcluster harboring dithiomethylamine, carbon monoxide, and cyanide ligands. This 2Fe subcluster is synthesized and inserted into [FeFe]-hydrogenase by three maturase enzymes denoted HydE, HydF, and HydG. HydE and HydG are radical S-adenosylmethionine enzymes and synthesize the nonprotein ligands of the H-cluster. HydF is a GTPase that functions as a scaffold or carrier for 2Fe subcluster production. Herein, we utilize UV-visible, circular dichroism, and electron paramagnetic resonance spectroscopic studies to establish the existence of redox active [4Fe-4S] and [2Fe-2S] clusters bound to HydF. We have used spectroelectrochemical titrations to assign iron-sulfur cluster midpoint potentials, have shown that HydF purifies with a reduced [2Fe-2S] cluster in the absence of exogenous reducing agents, and have tracked iron-sulfur cluster spectroscopic changes with quaternary structural perturbations. Our results provide an important foundation for understanding the maturation process by defining the iron-sulfur cluster content of HydF prior to its interaction with HydE and HydG. We speculate that the [2Fe-2S] cluster of HydF either acts as a placeholder for HydG-derived Fe(CO)2CN species or serves as a scaffold for 2Fe subcluster assembly.

[FeFe]-hydrogenase maturation: insights into the role HydE plays in dithiomethylamine biosynthesis.

HydE and HydG are radical S-adenosyl-l-methionine enzymes required for the maturation of [FeFe]-hydrogenase (HydA) and produce the nonprotein organic ligands characteristic of its unique catalytic cluster. The catalytic cluster of HydA (the H-cluster) is a typical [4Fe-4S] cubane bridged to a 2Fe-subcluster that contains two carbon monoxides, three cyanides, and a bridging dithiomethylamine as ligands. While recent studies have shed light on the nature of diatomic ligand biosynthesis by HydG, little information exists on the function of HydE. Herein, we present biochemical, spectroscopic, bioinformatic, and molecular modeling data that together map the active site and provide significant insight into the role of HydE in H-cluster biosynthesis. Electron paramagnetic resonance and UV-visible spectroscopic studies demonstrate that reconstituted HydE binds two [4Fe-4S] clusters and copurifies with S-adenosyl-l-methionine. Incorporation of deuterium from D2O into 5'-deoxyadenosine, the cleavage product of S-adenosyl-l-methionine, coupled with molecular docking experiments suggests that the HydE substrate contains a thiol functional group. This information, along with HydE sequence similarity and genome context networks, has allowed us to redefine the presumed mechanism for HydE away from BioB-like sulfur insertion chemistry; these data collectively suggest that the source of the sulfur atoms in the dithiomethylamine bridge of the H-cluster is likely derived from HydE's thiol containing substrate.

[FeFe]-hydrogenase maturation.

Hydrogenases are metalloenzymes that catalyze the reversible reduction of protons at unusual metal centers. This Current Topic discusses recent advances in elucidating the steps involved in the biosynthesis of the complex metal cluster at the [FeFe]-hydrogenase (HydA) active site, known as the H-cluster. The H-cluster is composed of a 2Fe subcluster that is anchored within the active site by a bridging cysteine thiolate to a [4Fe-4S] cubane. The 2Fe subcluster contains carbon monoxide, cyanide, and bridging dithiolate ligands. H-cluster biosynthesis is now understood to occur stepwise; standard iron-sulfur cluster assembly machinery builds the [4Fe-4S] cubane of the H-cluster, while three specific maturase enzymes known as HydE, HydF, and HydG assemble the 2Fe subcluster. HydE and HydG are both radical S-adenosylmethionine enzymes that interact with an iron-sulfur cluster binding GTPase scaffold, HydF, during the construction of the 2Fe subcluster moiety. In an unprecedented biochemical reaction, HydG cleaves tyrosine and decomposes the resulting dehydroglycine into carbon monoxide and cyanide ligands. The role of HydE in the biosynthetic pathway remains undefined, although it is hypothesized to be critical for the synthesis of the bridging dithiolate. HydF is the site where the complete 2Fe subcluster is formed and ultimately delivered to the immature hydrogenase protein in the final step of [FeFe]-hydrogenase maturation. This work addresses the roles of and interactions among HydE, HydF, HydG, and HydA in the formation of the mature [FeFe]-hydrogenase.

H-cluster assembly during maturation of the [FeFe]-hydrogenase.

The organometallic H-cluster at the active site of the [FeFe]-hydrogenase serves as the site of reversible binding and reduction of protons to produce H2. The H-cluster is unique in biology, and consists of a 2Fe subcluster tethered to a typical [4Fe-4S] cluster by a single cysteine ligand. The remaining ligands to the 2Fe subcluster include three carbon monoxides, two cyanides, and a dithiomethylamine. This mini-review will focus on the significant advances in recent years in understanding the pathway for H-cluster biosynthesis, as well as the structures, roles, and mechanisms of the three enzymes directly involved.