The [4Fe–4S]-cluster coordination of [FeFe]-hydrogenase maturation protein HydF as revealed by EPR and HYSCORE spectroscopies

Abstract

[FeFe] hydrogenases are key enzymes for bio(photo)production of molecular hydrogen, and several efforts are underway to understand how their complex active site is assembled. This site contains a [4Fe-4S]-2Fe cluster and three conserved maturation proteins are required for its biosynthesis. Among them, HydF has a double task of scaffold, in which the dinuclear iron precursor is chemically modified by the two other maturases, and carrier to transfer this unit to a hydrogenase containing a preformed [4Fe-4S]-cluster. This dual role is associated with the capability of HydF to bind and dissociate an iron-sulfur center, due to the presence of the conserved FeS-cluster binding sequence CxHx(46-53)HCxxC. The recently solved three-dimensional structure of HydF from Thermotoga neapolitana described the domain containing the three cysteines which are supposed to bind the FeS cluster, and identified the position of two conserved histidines which could provide the fourth iron ligand. The functional role of two of these cysteines in the activation of [FeFe]-hydrogenases has been confirmed by site-specific mutagenesis. On the other hand, the contribution of the three cysteines to the FeS cluster coordination sphere is still to be demonstrated. Furthermore, the potential role of the two histidines in [FeFe]-hydrogenase maturation has never been addressed, and their involvement as fourth ligand for the cluster coordination is controversial. In this work we combined site-specific mutagenesis with EPR (electron paramagnetic resonance) and HYSCORE (hyperfine sublevel correlation spectroscopy) to assign a role to these conserved residues, in both cluster coordination and hydrogenase maturation/activation, in HydF proteins from different microorganisms.

[FeFe]- and [NiFe]-hydrogenase diversity, mechanism, and maturation

“…The observation and significance of [2Fe-2S] + cluster binding by HydF is still an open issue. While similar overlapping axial signals to those initially reported [241] have now been observed in reduced preparations of both Ca and Tn samples, it has been suggested this signal arises from a radical in close proximity to the [4Fe-4S] + cluster [248,249,251]. Regardless, multiple lines of evidence support the notion that the 2Fe subcluster bound by HydF resembles the H-cluster, and may potentially even be directly linked to the [4Fe-4S] cubane present on HydF [245,251].…”

“…The coordination environment for the [4Fe-4S] cluster utilizes three conserved cysteine residues; the fourth coordination site appears to be variable, either being a histidine in the case of Ca HydF or a labile/ exchangeable ligand in Thermotoga maritima (Tm) and Thermotoga neopolitana (Tn) HydF [242,246,[248][249][250]. The observation and significance of [2Fe-2S] + cluster binding by HydF is still an open issue.…”

“…The FeS cluster states of both wild type and variant forms of HydF have been examined from various species and all of the results provide evidence for [4Fe-4S] cluster binding to HydF [237,241,[244][245][246][247][248][249]. The coordination environment for the [4Fe-4S] cluster utilizes three conserved cysteine residues; the fourth coordination site appears to be variable, either being a histidine in the case of Ca HydF or a labile/ exchangeable ligand in Thermotoga maritima (Tm) and Thermotoga neopolitana (Tn) HydF [242,246,[248][249][250].…”

Advanced paramagnetic resonance spectroscopies of iron–sulfur proteins: Electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM)

“…While the advanced EPR spectroscopic characterization of hydrogenase has provided an abundance of information including ligand unpaired spin density, [111] thiolate bridge atom identification, [112–115], cluster assembly, [113, 116–120] [121] and model complexes, [122–124] all extensively reviewed elsewhere, [108, 116, 125] we will briefly show the particular example of the 57 Fe ENDOR and HYSCORE work of the [4Fe4S] cluster of the [FeFe]-hydrogenase. [41] This study determined that the Fe p of the paramagnetic [Fe 1+ –Fe 2+ ] oxidized H-center is in the Fe I oxidation state and binds to the cuboidal [4Fe4S] cluster, while the distal iron, Fe d , alternates between Fe I (reduction) and Fe II (oxidation) states.…”

Metallocofactor assembly for [FeFe]-hydrogenases

“…Given the important role for HydF in the complex process of [2Fe] H subcluster assembly, it is perhaps unsurprising that the reported nature of the cluster(s) bound to HydF vary depending on the protein source, chemical reconstitution, heterologous or homologous expression, and co-expression with or without the other maturases (termed HydF EG and HydF ΔEG respectively). There is good agreement that HydF binds a [4Fe-4S] cluster, but variability in the observed state of the second Fe-S cluster: for example, reconstituted TmHydF ΔEG and TnHydF ΔEG likely bear no second cluster [37,38], EPR spectra of unreconstituted CaHydF ΔEG shows a signal resembling a [2Fe-2S] cluster [39,40] and IR spectroscopy suggests multiple diatomic ligands bound to CaHydF EG [40,41]. Models for cluster assembly by HydF agree that the [4Fe-4S] is assembled by housekeeping iron sulfur cluster biosynthetic enzymes.…”