Structural differences of oxidized iron-sulfur and nickel-iron cofactors in O2-tolerant and O2-sensitive hydrogenases studied by X-ray absorption spectroscopy.

The class of [NiFe]-hydrogenases comprises oxygen-sensitive periplasmic (PH) and oxygen-tolerant membrane-bound (MBH) enzymes. For three PHs and four MBHs from six bacterial species, structural features of the nickel-iron active site of hydrogen turnover and of the iron-sulfur clusters functioning in electron transfer were determined using X-ray absorption spectroscopy (XAS). Fe-XAS indicated surplus oxidized iron and a lower number of ~2.7 Å Fe-Fe distances plus additional shorter and longer distances in the oxidized MBHs compared to the oxidized PHs. This supported a double-oxidized and modified proximal FeS cluster in all MBHs with an apparent trimer-plus-monomer arrangement of its four iron atoms, in agreement with crystal data showing a [4Fe3S] cluster instead of a [4Fe4S] cubane as in the PHs. Ni-XAS indicated coordination of the nickel by the thiol group sulfurs of four conserved cysteines and at least one iron-oxygen bond in both MBH and PH proteins. Structural differences of the oxidized inactive [NiFe] cofactor of MBHs in the Ni-B state compared to PHs in the Ni-A state included a ~0.05 Å longer Ni-O bond, a two times larger spread of the Ni-S bond lengths, and a ~0.1 Å shorter Ni-Fe distance. The modified proximal [4Fe3S] cluster, weaker binding of the Ni-Fe bridging oxygen species, and an altered localization of reduced oxygen species at the active site may each contribute to O2 tolerance.

How oxygen attacks [FeFe] hydrogenases from photosynthetic organisms.

Green algae such as Chlamydomonas reinhardtii synthesize an [FeFe] hydrogenase that is highly active in hydrogen evolution. However, the extreme sensitivity of [FeFe] hydrogenases to oxygen presents a major challenge for exploiting these organisms to achieve sustainable photosynthetic hydrogen production. In this study, the mechanism of oxygen inactivation of the [FeFe] hydrogenase CrHydA1 from C. reinhardtii has been investigated. X-ray absorption spectroscopy shows that reaction with oxygen results in destruction of the [4Fe-4S] domain of the active site H-cluster while leaving the di-iron domain (2Fe(H)) essentially intact. By protein film electrochemistry we were able to determine the order of events leading up to this destruction. Carbon monoxide, a competitive inhibitor of CrHydA1 which binds to an Fe atom of the 2Fe(H) domain and is otherwise not known to attack FeS clusters in proteins, reacts nearly two orders of magnitude faster than oxygen and protects the enzyme against oxygen damage. These results therefore show that destruction of the [4Fe-4S] cluster is initiated by binding and reduction of oxygen at the di-iron domain-a key step that is blocked by carbon monoxide. The relatively slow attack by oxygen compared to carbon monoxide suggests that a very high level of discrimination can be achieved by subtle factors such as electronic effects (specific orbital overlap requirements) and steric constraints at the active site.

[NiFe] and [FeS] cofactors in the membrane-bound hydrogenase of Ralstonia eutropha investigated by X-ray absorption spectroscopy: insights into O(2)-tolerant H(2) cleavage.

Molecular features that allow certain [NiFe] hydrogenases to catalyze the conversion of molecular hydrogen (H(2)) in the presence of dioxygen (O(2)) were investigated. Using X-ray absorption spectroscopy (XAS), we compared the [NiFe] active site and FeS clusters in the O(2)-tolerant membrane-bound hydrogenase (MBH) of Ralstonia eutropha and the O(2)-sensitive periplasmic hydrogenase (PH) of Desulfovibrio gigas. Fe-XAS indicated an unusual complement of iron-sulfur centers in the MBH, likely based on a specific structure of the FeS cluster proximal to the active site. This cluster is a [4Fe4S] cubane in PH. For MBH, it comprises less than ~2.7 Å Fe-Fe distances and additional longer vectors of ≥3.4 Å, consistent with an Fe trimer with a more isolated Fe ion. Ni-XAS indicated a similar architecture of the [NiFe] site in MBH and PH, featuring Ni coordination by four thiolates of conserved cysteines, i.e., in the fully reduced state (Ni-SR). For oxidized states, short Ni-µO bonds due to Ni-Fe bridging oxygen species were detected in the Ni-B state of the MBH and in the Ni-A state of the PH. Furthermore, a bridging sulfenate (CysSO) is suggested for an inactive state (Ni(ia)-S) of the MBH. We propose that the O(2) tolerance of the MBH is mainly based on a dedicated electron donation from a modified proximal FeS cluster to the active site, which may favor formation of the rapidly reactivated Ni-B state instead of the slowly reactivated Ni-A state. Thereby, the catalytic activity of the MBH is facilitated in the presence of both H(2) and O(2).

Protein-protein complex formation affects the Ni-Fe and Fe-S centers in the H2-sensing regulatory hydrogenase from Ralstonia eutropha H16.

The regulatory Ni-Fe hydrogenase (RH) from the H(2)-oxidizing bacterium Ralstonia eutropha functions as an oxygen-resistant hydrogen sensor, which is composed of the large, active-site-containing HoxC subunit and the small subunit HoxB carrying Fe-S clusters. In vivo, the HoxBC subunits form a dimer designated as RH(wt). The RH(wt) protein transmits its signals to the histidine protein kinase HoxJ, which itself forms a homotetramer and a stable complex with RH(wt) (RH(wt)-HoxJ(wt)), located in the cytoplasm. In this study, we used X-ray absorption (XAS), electron paramagnetic resonance (EPR), and Fourier transform infrared (FTIR) spectroscopy to investigate the impact of various complexes between RH and HoxJ on the structural and electronic properties of the Ni-Fe active site and the Fe-S clusters. Aside from the RH(wt) protein and the RH(wt)-HoxJ(wt) complex, we investigated the RH(stop) protein, which consists of only one HoxB and HoxC unit due to the missing C-terminus of HoxB, as well as RH(wt)-HoxJ(Deltakinase), in which the histidine protein kinase lacks the transmitter domain. All constructs reacted with H(2), leading to the formation of the EPR-detectable Ni(III)-C state of the active site and to the reduction of Fe-S clusters detectable by XAS, thus corroborating that H(2) cleavage is independent of the presence of the HoxJ protein. In RH(stop), presumably one Fe-S cluster was lost during the preparation procedure. The coordination of the active site Ni in RH(stop) differed from that in RH(wt) and the RH(wt)-HoxJ complexes, in which additional Ni--O bonds were detected by XAS. The Ni--O bonds caused only very minor changes of the EPR g-values of the Ni-C and Ni-L states and of the IR vibrational frequencies of the diatomic CN(-) and CO ligands at the active-site Fe ion. Both one Fe-S cluster in HoxB and an oxygen-rich Ni coordination seem to be stabilized by RH dimerization involving the C-terminus of HoxB and by complex formation with HoxJ.

The structure of the active site H-cluster of [FeFe] hydrogenase from the green alga Chlamydomonas reinhardtii studied by X-ray absorption spectroscopy.

The [FeFe] hydrogenase (CrHydA1) of the green alga Chlamydomonas reinhardtii is the smallest hydrogenase known and can be considered as a "minimal unit" for biological H(2) production. Due to the absence of additional FeS clusters as found in bacterial [FeFe] hydrogenases, it was possible to specifically study its catalytic iron-sulfur cluster (H-cluster) by X-ray absorption spectroscopy (XAS) at the Fe K-edge. The XAS analysis revealed that the CrHydA1 H-cluster consists of a [4Fe4S] cluster and a diiron site, 2Fe(H), which both are similar to their crystallographically characterized bacterial counterparts. Determination of the individual Fe-Fe distances in the [4Fe4S] cluster ( approximately 2.7 A) and in the 2Fe(H) unit ( approximately 2.5 A) was achieved. Fe-C( horizontal lineO/N) and Fe-S bond lengths were in good agreement with crystallographic data on bacterial enzymes. The loss of Fe-Fe distances in protein purified under mildly oxidizing conditions indicated partial degradation of the H-cluster. Bond length alterations detected after incubation of CrHydA1 with CO and H(2) were related to structural and oxidation state changes at the catalytic Fe atoms, e.g., to the binding of an exogenous CO at 2Fe(H) in CO-inhibited enzyme. Our XAS studies pave the way for the monitoring of atomic level structural changes at the H-cluster during H(2) catalysis.

Introduction of methionines in the gas channel makes [NiFe] hydrogenase aero-tolerant.

Hydrogenases catalyze the conversion between 2H(+) + 2e(-) and H(2)(1). Most of these enzymes are inhibited by O(2), which represents a major drawback for their use in biotechnological applications. Improving hydrogenase O(2) tolerance is therefore a major contemporary challenge to allow the implementation of a sustainable hydrogen economy. We succeeded in improving O(2) tolerance, which we define here as the ability of the enzyme to resist for several minutes to O(2) exposure, by substituting with methionines small hydrophobic residues strongly conserved in the gas channel. Remarkably, the mutated enzymes remained active in the presence of an O(2) concentration close to that found in aerobic solutions in equilibrium with air, while the wild type enzyme is inhibited in a few seconds. Crystallographic and spectroscopic studies showed that the structure and the chemistry at the active site are not affected by the mutations. Kinetic studies demonstrated that the inactivation is slower and reactivation faster in these mutants. We propose that in addition to restricting O(2) diffusion to the active site of the enzyme, methionine may also interact with bound peroxide and provide an assisted escape route for H(2)O(2) toward the gas channel. These results show for the first time that it is possible to improve O(2)-tolerance of [NiFe] hydrogenases, making possible the development of biohydrogen production systems.

The [FeFe]-hydrogenase maturation protein HydF contains a H-cluster like [4Fe4S]-2Fe site.

Formation of the catalytic six-iron complex (H-cluster) of [FeFe]-hydrogenase (HydA) requires its interaction with a specific maturation protein, HydF. Comparison by X-ray absorption spectroscopy at the Fe K-edge of HydF from Clostridium acetobutylicum and HydA1 from Chlamydomonas reinhardtii revealed that the overall structure of the iron site in both proteins is highly similar, comprising a [4Fe4S] cluster (Fe-Fe distances of ∼2.7Å) and a di-iron unit (Fe-Fe distance of ∼2.5Å). Thus, a precursor of the whole H-cluster is assembled on HydF. Formation of the core structures of both the 4Fe and 2Fe units may require only the housekeeping [FeS] cluster assembly machinery of the cell. Presumably, only the 2Fe cluster is transferred from HydF to HydA1, thereby forming the active site.

Redox intermediates of the Mn-Fe Site in subunit R2 of Chlamydia trachomatis ribonucleotide reductase: an X-ray absorption and EPR study.

The R2 protein of class I ribonucleotide reductase (RNR) from Chlamydia trachomatis (Ct) can contain a Mn-Fe instead of the standard Fe-Fe cofactor. Ct R2 has a redox-inert phenylalanine replacing the radical-forming tyrosine of classic RNRs, which implies a different mechanism of O(2) activation. We studied the Mn-Fe site by x-ray absorption spectroscopy (XAS) and EPR. Reduced R2 in the R1R2 complex (R2(red)) showed an isotropic six-line EPR signal at g approximately 2 of the Mn(II)Fe(II) state. In oxidized R2 (R2(ox)), the Mn(III)Fe(III) state exhibited EPR g values of 2.013, 2.009, and 2.015. By XAS, Mn-Fe distances and oxidation states of intermediates were determined and assigned as follows: approximately 4.15 A, Mn(II)Fe(II); approximately 3.25 A, Mn(III)Fe(II); approximately 2.90 A, Mn(III)Fe(III); and approximately 2.75 A, Mn(IV)Fe(III). Shortening of the Mn/Fe-ligand bond lengths indicated formation of additional metal bridges, i.e. microO(H) and/or peroxidic species, upon O(2) activation at the site. The structural parameters suggest overall configurations of the Mn-Fe site similar to those of homo-metallic sites in other R2 proteins. However, the approximately 2.90 A and approximately 2.75 A Mn-Fe distances, typical for di-microO(H) metal bridging, are shorter than inter-metal distances in any R2 crystal structure. In diffraction data collection, such bridges may be lost due to rapid x-ray photoreduction of high-valent metal ions, as demonstrated here for Fe(III) by XAS.