Protein–Protein Complex Formation Affects the Ni–Fe and Fe–S Centers in the H<sub>2</sub>‐Sensing Regulatory Hydrogenase from <i>Ralstonia eutropha</i> H16

Löscher, Simone; Gebler, Antje; Stein, Matthias; Sanganas, Oliver; Buhrke, Thorsten; Zebger, Ingo; Dau, Holger; Friedrich, Bärbel; Lenz, Oliver; Haumann, Michael

doi:10.1002/cphc.200901007

Cited by 12 publications

(26 citation statements)

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“…The results in the present study support the previously made conclusion (11,43,45,54 (24,43,85 (53) and of the membrane-bound type (MBH) from R. eutropha (25,31). Two CN -and one CO at the active site iron were inferred by FTIR (43).…”

Section: Resultssupporting

confidence: 91%

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[NiFe] and [FeS] Cofactors in the Membrane-Bound Hydrogenase of Ralstonia eutropha Investigated by X-ray Absorption Spectroscopy: Insights into O₂-Tolerant H₂ Cleavage

et al. 2011

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Molecular features that allow certain [NiFe] hydrogenases to catalyze the conversion of molecular hydrogen (H2) in the presence of dioxygen (O2) were investigated. Using X-ray absorption spectroscopy (XAS), we compared the [NiFe] active site and FeS clusters in the O2-tolerant membrane-bound hydrogenase (MBH) of Ralstonia eutropha and the O2-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 (Niia-S) of the MBH. We propose that the O2 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 H2 and O2.

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Section: Resultssupporting

confidence: 91%

“…3). In the oxidized MBH at pH 8.0, a pronounced increase of the primary edge maximum at ~8350 eV suggested additional terminal oxygen ligands at the Ni(II) (23,58,82,85) in the Ni ia -S state and therefore a substantially different site structure compared to Ni-B.…”

Section: Resultsmentioning

confidence: 99%

[NiFe] and [FeS] Cofactors in the Membrane-Bound Hydrogenase of Ralstonia eutropha Investigated by X-ray Absorption Spectroscopy: Insights into O₂-Tolerant H₂ Cleavage

et al. 2011

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“…In the absence of H 2 , HoxJ transfers phosphate to the transcriptional response regulator HoxA, which is inactive in the phosphorylated form. Biochemical and genetic data suggest that signal transduction between the regulatory [NiFe]-hydrogenase and HoxJ involves an electron transport process (80)(81)(82)(83)(84)(85)(86). The regulatory [NiFe]-hydrogenase contains an active site similar to that of the standard [NiFe]-hydrogenases.…”

Section: Discussionmentioning

confidence: 99%

Hydrogen Formation and Its Regulation in Ruminococcus albus: Involvement of an Electron-Bifurcating [FeFe]-Hydrogenase, of a Non-Electron-Bifurcating [FeFe]-Hydrogenase, and of a Putative Hydrogen-Sensing [FeFe]-Hydrogenase

et al. 2014

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bRuminococcus albus 7 has played a key role in the development of the concept of interspecies hydrogen transfer. The rumen bacterium ferments glucose to 1.3 acetate, 0.7 ethanol, 2 CO 2 , and 2.6 H 2 when growing in batch culture and to 2 acetate, 2 CO 2 , and 4 H 2 when growing in continuous culture in syntrophic association with H 2 -consuming microorganisms that keep the H 2 partial pressure low. The organism uses NAD ؉ and ferredoxin for glucose oxidation to acetyl coenzyme A (acetyl-CoA) and CO 2 , NADH for the reduction of acetyl-CoA to ethanol, and NADH and reduced ferredoxin for the reduction of protons to H 2 . Of all the enzymes involved, only the enzyme catalyzing the formation of H 2 from NADH remained unknown. Here, we report that R. T he genus Ruminococcus (class Clostridia) consists of species of anaerobic, Gram-positive bacteria. One or more species of this genus are found in significant numbers in the intestines of humans (enterotype 3) (1, 2) and in the rumen and colon of herbivores (3, 4). Ruminococcus flavefaciens and Ruminococcus albus are among the most important plant cell wall-degrading bacteria in the rumen. These two species produce all required enzymes for hydrolyzing the plant cell wall polysaccharides, cellulose and hemicellulose (5, 6).R. albus was isolated in 1957 from the rumen of cattle by Hungate (7,8), who showed that in batch culture the bacterium ferments cellulose, cellobiose, or glucose to acetate, CO 2 , ethanol, and H 2 . Nevertheless, neither H 2 nor ethanol accumulate to high concentrations in the rumen (9, 10). The low H 2 concentrations in the rumen have been explained by the presence of H 2 -consuming microorganisms, such as Methanobrevibacter ruminantium (formerly Methanobacterium ruminantium), that consume H 2 more rapidly than it is formed from cellulose by R. albus (11). The laboratory of Bryant and Wolin then showed in 1973 that the fermentation of glucose by R. albus strain 7 was shifted from 1.3 acetic acid, 0.7 ethanol, 2 CO 2 , and 2.6 H 2 in batch culture to 2 acetic acid, 2 CO 2 , and 4 H 2 in chemostat culture together with Wolinella succinogenes (formerly Vibrio succinogenes), which grew on H 2 and fumarate producing succinate, keeping the H 2 concentration low (12). The interspecies cooperation allowed W. succinogenes to grow at the expense of H 2 produced by R. albus, but it also offered an energetic advantage to R. albus in the form of a higher ATP gain (4 mol of ATP instead of 3.3 mol of glucose fermented) and, consequently, a better growth yield (13). At low H 2 partial pressures, the free energy associated with glucose fermentation is more negative than that at high H 2 partial pressures, which is the thermodynamic basis for the different ATP gains (13). The fermentation of R. albus on glucose has been modeled (14). The literature on interspecies H 2 and formate transfer has been reviewed recently (15).Enzymatic analyses have revealed that R. albus strain 7 grown in batch culture on glucose contains an NAD-specific glyceraldehyde-3-phosphate dehydr...

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“…by genetic engineering of the protein). Such attempts are opposed to the narrowing of O 2 -conducting gas channels (6,71), which seems to provide O 2 tolerance in H 2 sensor proteins (3,72,73). In the case of membrane-bound [NiFe]-H 2 ases, an altered structure and redox potential of the FeS cluster proximal to the active site is the key to O 2 tolerance (64,70,74,75).…”

Section: Discussionmentioning

confidence: 99%

O2 Reactions at the Six-iron Active Site (H-cluster) in [FeFe]-Hydrogenase

Lambertz

Leidel

Havelius

et al. 2011

Journal of Biological Chemistry

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144

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[FeFe]-hydrogenase (H 2 ase) 4 proteins are the most active biological catalysts for the production of molecular hydrogen (H 2 ) from proton reduction, with reported turnover rates of up to 10 4 s Ϫ1 (1, 2). Therefore, these enzymes are of high interest for biotechnology, aiming at the generation of H 2 as a renewable fuel (1, 3-5). However, a severe limitation for such applications is the rapid inactivation of [FeFe]-H 2 ases by dioxygen (O 2 ) (6, 7). Understanding the mechanism of O 2 -induced inactivation may allow the improvement of enzyme features to yield increased O 2 tolerance (e.g. by genetic engineering, which has already been demonstrated for NiFe hydrogenases) (8 -10).[FeFe]-H 2 ases are found in certain bacteria and green algae (11, 12). All of these enzymes contain an active site that consists of an inorganic iron complex, which is denoted as the H-cluster (13-15). The [FeFe]-H 2 ase enzymes from anaerobic bacteria in addition bind several iron-sulfur (FeS) clusters, serving as a relay for electron transfer to and from the active site (13-15).[FeFe]-H 2 ases from green algae represent the minimal unit for biological H 2 production because they contain only the H-cluster, whereas accessory FeS clusters are absent (16). This feature renders these enzymes most suitable for spectroscopic investigations on O 2 -induced inactivation (17), focusing on the active site reactions. The general structure of the H-cluster has been unraveled by crystallography, x-ray absorption spectroscopy (XAS), EPR, and FTIR spectroscopy on H 2 ase protein from various organisms (18). The H-cluster structure in both bacteria and green algae appears to be similar overall (16,19,20). It features a [4Fe4S] cubane cluster, which is bound by four cysteine residues to the protein and is linked by one of them to a binuclear iron unit (2Fe H ) (Fig. 1). The latter carries two cyanide (CN Ϫ ) ligands and three carbon monoxide (CO) ligands (21, 22) and presumably an azadithiolate group (adt; (SCH 2 ) 2 NH) in the metal-bridging position (21,23,24). Both types of diatomic ligands are probably derived from a biosynthetic pathway starting with tyrosine (25-27). The nitrogen atom of the adt has been proposed to be involved in proton transfer at the active site (21, 28). The H-cluster structure is assembled in a complex reaction involving three maturation proteins (29 -34). H 2 formation has been proposed to involve the binding and reduction of a single proton, which transiently creates a hydride ligand, either located in a bridging position between the two iron ions or terminally bound at the distal iron ion of the 2Fe H moiety (Fig. 1). After a second protonation step, H 2 is released from the H-cluster (35)(36)(37) 4 The abbreviations used are: H 2 ase, hydrogenase; adt, azadithiolate; EXAFS, extended x-ray absorption fine structure; NaDT, sodium dithionite; ROS, reactive oxygen species; XANES, x-ray absorption near edge structure; XAS, x-ray absorption spectroscopy; FT, Fourier transform.

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Protein–Protein Complex Formation Affects the Ni–Fe and Fe–S Centers in the H₂‐Sensing Regulatory Hydrogenase from Ralstonia eutropha H16

Cited by 12 publications

References 51 publications

[NiFe] and [FeS] Cofactors in the Membrane-Bound Hydrogenase of Ralstonia eutropha Investigated by X-ray Absorption Spectroscopy: Insights into O₂-Tolerant H₂ Cleavage

[NiFe] and [FeS] Cofactors in the Membrane-Bound Hydrogenase of Ralstonia eutropha Investigated by X-ray Absorption Spectroscopy: Insights into O₂-Tolerant H₂ Cleavage

Hydrogen Formation and Its Regulation in Ruminococcus albus: Involvement of an Electron-Bifurcating [FeFe]-Hydrogenase, of a Non-Electron-Bifurcating [FeFe]-Hydrogenase, and of a Putative Hydrogen-Sensing [FeFe]-Hydrogenase

O2 Reactions at the Six-iron Active Site (H-cluster) in [FeFe]-Hydrogenase

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Protein–Protein Complex Formation Affects the Ni–Fe and Fe–S Centers in the H2‐Sensing Regulatory Hydrogenase from Ralstonia eutropha H16

Cited by 12 publications

References 51 publications

[NiFe] and [FeS] Cofactors in the Membrane-Bound Hydrogenase of Ralstonia eutropha Investigated by X-ray Absorption Spectroscopy: Insights into O2-Tolerant H2 Cleavage

[NiFe] and [FeS] Cofactors in the Membrane-Bound Hydrogenase of Ralstonia eutropha Investigated by X-ray Absorption Spectroscopy: Insights into O2-Tolerant H2 Cleavage

Hydrogen Formation and Its Regulation in Ruminococcus albus: Involvement of an Electron-Bifurcating [FeFe]-Hydrogenase, of a Non-Electron-Bifurcating [FeFe]-Hydrogenase, and of a Putative Hydrogen-Sensing [FeFe]-Hydrogenase

O2 Reactions at the Six-iron Active Site (H-cluster) in [FeFe]-Hydrogenase

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Protein–Protein Complex Formation Affects the Ni–Fe and Fe–S Centers in the H₂‐Sensing Regulatory Hydrogenase from Ralstonia eutropha H16

[NiFe] and [FeS] Cofactors in the Membrane-Bound Hydrogenase of Ralstonia eutropha Investigated by X-ray Absorption Spectroscopy: Insights into O₂-Tolerant H₂ Cleavage

[NiFe] and [FeS] Cofactors in the Membrane-Bound Hydrogenase of Ralstonia eutropha Investigated by X-ray Absorption Spectroscopy: Insights into O₂-Tolerant H₂ Cleavage