Electronic structure of the unique [4Fe-3S] cluster in O ₂ -tolerant hydrogenases characterized by ⁵⁷ Fe Mössbauer and EPR spectroscopy

Abstract

Iron-sulfur clusters are ubiquitous electron transfer cofactors in hydrogenases. Their types and redox properties are important for H 2 catalysis, but, recently, their role in a protection mechanism against oxidative inactivation has also been recognized for a [4Fe-3S] cluster in O 2 -tolerant group 1 [NiFe] hydrogenases. This cluster, which is uniquely coordinated by six cysteines, is situated in the proximity of the catalytic [NiFe] site and exhibits unusual redox versatility. The [4Fe-3S] cluster in hydrogenase (Hase) I from Aquifex aeolicus performs two redox transitions within a very small potential range, forming a superoxidized state above +200 mV vs. standard hydrogen electrode (SHE). Crystallographic data has revealed that this state is stabilized by the coordination of one of the iron atoms to a backbone nitrogen. Thus, the proximal [4Fe-3S] cluster undergoes redox-dependent changes to serve multiple purposes beyond classical electron transfer. In this paper, we present fielddependent Recently, a subclass of O 2 -tolerant, membrane-bound hydrogenases (MBH) was recognized to possess an uncommon proximal iron-sulfur cluster with unique redox properties that enable a protection mechanism against oxidative inactivation of the [NiFe] site. Typical representatives of such hydrogenases are those from Aquifex aeolicus (3), Hydrogenovibrio marinus (4), Ralstonia eutropha (5), and Escherichia coli (6). Three independent crystal structure investigations (4, 5, 7) revealed that the proximal cluster in these enzymes is not a classical [4Fe-4S] but, instead, a [4Fe-3S] cluster with an unusual six-cysteine binding motif (CXCCX 94 CX 4 CX 28 C) that is unique for the subclass of O 2 -tolerant enzymes (8, 9) (SI Appendix, Fig. S1). Four of the six cysteine residues (C) coordinate the cluster in the usual manner as terminal ligands to the [4Fe-3S] core, whereas the sulfur (S − ) atom of one of the supernumerary cysteines (C) substitutes for an inorganic sulfide (S 2− ) in the cubane core, and the other serves as a second terminal ligand to one of the Fe atoms (4, 5, 7) (Fig. 1). In contrast, the structures of the medial and distal iron-sulfur clusters and their arrangement relative to the [NiFe] site are almost identical to those of standard hydrogenases (1,4,5).Conventional [4Fe-4S] clusters perform either a single-electron 1+/2+ redox transition (in low-potential ferredoxins) or a 2+/3+ transition in high-potential iron-sulfur proteins (HiPIPs) (2). Both forms share the same isoelectronic 2+ oxidation state with spin S = 0, albeit with some minor differences in structures and electron delocalization (10, 11). In contrast, the proximal [4Fe-3S] cluster in hydrogenase (Hase) I from A. aeolicus can attain three redox states within a redox potential span of only 150 mV, two of which are paramagnetic with S = 1/2, as we could show by EPR-detected potentiometric titrations (3). Particularly, a superoxidized state, not known for low-potential [4Fe-4S] clusters (12), manifests at high potentials (3). According to the cry...

Models of the Ni-L and Ni-SIa States of the [NiFe]-Hydrogenase Active Site

“…20 Mössbauer data for the [NiFe]-hydrogenases are sparse and suffer from difficulties in identifying and correcting for the dominating subspectra of the accessory FeS clusters. 37,38 Isomer shifts in the range of 0.05–0.15 mm s −1 have been assigned to the iron center of the [NiFe] center. 39 …”

Multiscale Simulations Give Insight into the Hydrogen In and Out Pathways of [NiFe]-Hydrogenases from Aquifex aeolicus and Desulfovibrio fructosovorans

“…Different techniques such as spectroscopy, 18,97,98 site-directed mutagenesis, 67,92 and in silico approaches 68,69,82−84 have been used to clarify the complexity associated with this family.…”

Biohydrogen for a New Generation of H2/O2 Biofuel Cells: A Sustainable Energy Perspective

“…[105] All the O 2 -tolerant Hases share the same phenotypes in addition to O 2 tolerance: electron transfer from the [NiFe] active site to a membrane-bound diheme cytochrome b through a conductive line of three FeS clusters (electrons are then transferred via a quinone pool to a bc cytochrome then to a cytochrome oxidase) [96] ( Figure 5 A), CO-insensitivity (in contrast to O 2 -sensitive Hases), and higher redox potentials of the FeS clusters than the clusters in O 2 -sensitive Hases (Table 2). [77,78,[106][107][108][109][110][111][112] In addition, because it is purified from a hyperthermophilic bacterium, Aa MbH is able to oxidize H 2 over a temperature range from 25 to 85 8C. [106] The O 2 -tolerant Hases are also much worse H 2 producers than O 2 -sensitive Hases.…”