Reduction of Unusual Iron-Sulfur Clusters in the H2-sensing Regulatory Ni-Fe Hydrogenase from Ralstonia eutropha H16

Buhrke, Thorsten; Löscher, Simone; Lenz, Oliver; Schlodder, E.; Zebger, Ingo; Andersen, Lars Klembt; Hildebrandt, Peter; Meyer‐Klaucke, Wolfram; Dau, Holger; Friedrich, Bärbel; Haumann, Michael

doi:10.1074/jbc.m500601200

Cited by 47 publications

(99 citation statements)

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“…Spectra were collected maximally for a scan range of 6,950 -8,450 eV. Deadtime-corrected XAS spectra were averaged after energy calibration of each scan by using the peak at 7,112 eV in the 1 st derivative of the absorption spectrum of an Fe-foil as an energy standard (estimated accuracy Ϯ 0.1 eV) (28,29). Data were then normalized, and extended EXAFS oscillations were extracted (39).…”

Section: Methodsmentioning

confidence: 99%

“…K␣-fluorescence-detected XAS spectra at the Fe K-edge were collected at T ϭ 20 K using an energy-resolving 13-element Ge detector and a helium cryostat as previously described (28,29) at beamline D2 of the EMBL outstation (at HASYLAB, DESY). Harmonic rejection was achieved by detuning of the Si (111) double-crystal monochromator to 50% of its peak intensity.…”

Section: Methodsmentioning

confidence: 99%

“…The shape of the Fe K-edge spectrum of H red (Fig. 4A) reveals a predominant coordination of the Fe atoms by the S atoms of the conserved cysteine residues in CrHydA1 (25); indications for O-ligation to Fe are absent (28,29). The increased primary K-edge maximum (25).…”

Section: Composition (ϸ02 MM In Solution) As Displayed Inmentioning

confidence: 99%

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How oxygen attacks [FeFe] hydrogenases from photosynthetic organisms

Stripp

Goldet

Brandmayr

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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323

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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 (2FeH) 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 2FeH 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.EXAFS ͉ H-cluster ͉ protein film electrochemistry ͉ biological hydrogen production ͉ green algae

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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How oxygen attacks [FeFe] hydrogenases from photosynthetic organisms

Stripp

Goldet

Brandmayr

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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323

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“…The resulting unready, inactive enzyme requires a long term reactivation with H 2 to acquire a catalytically active conformation. Two other hydrogenases in R. eutropha, the soluble, hexameric NAD ϩ -reducing hydrogenase and the cytoplasmic H 2 -sensing hydrogenase, share the exceptional ability of being quickly reactivated after exposure to oxygen, and for none of them have Ni u -A signals been detected (25,61).…”

Section: Ftir Spectroscopic Characterization Of the Mbh Protein As A mentioning

confidence: 99%

Spectroscopic Insights into the Oxygen-tolerant Membrane-associated [NiFe] Hydrogenase of Ralstonia eutropha H16

Saggu

Zebger

Ludwig

et al. 2009

Journal of Biological Chemistry

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112

209

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Hydrogenases are metalloenzymes that catalyze the reversible cleavage of H 2 into protons and electrons and play a pivotal role in the energy metabolism of many microorganisms (1). They are grouped into three phylogenetically distinct classes as follows: the di-iron [FeFe], nickel-iron [NiFe], and iron-sulfur cluster-free [Fe] hydrogenases (2-6). The basic module of [NiFe] hydrogenases consists of two subunits, a large subunit that contains the [NiFe] active site and a small subunit that accommodates one to three electron-transferring iron-sulfur

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“…For this study, we select the nickel-iron regulatory hydrogenase (RH) from the 'Knallgas' bacterium Ralstonia eutropha. IR studies have shown the active site of the RH to harbour a standard set of diatomic ligands on the Fe, 17 18,19 The Ni-S state is assigned as Ni II /Fe II and is EPR-silent. 17 Proton-coupled 1-electron reduction leads to the Ni-C state, which is detectable by EPR and is assigned as Ni III /Fe II with a bridging H À (Fig.…”

mentioning

confidence: 99%

Attenuated total reflectance infrared spectroelectrochemistry at a carbon particle electrode; unmediated redox control of a [NiFe]-hydrogenase solution

Healy

Ash

Lenz

et al. 2013

Phys. Chem. Chem. Phys.

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We report a versatile infrared spectroscopic method for studying redox chemistry of metalloproteins, and demonstrate for the first time electrochemically-induced changes to the active site of the regulatory [NiFe]-hydrogenase from Ralstonia eutropha. A carbon particle network working electrode allows control over a wide potential window without the need for solution mediators.Hydrogenases are able to oxidise or produce H 2 at high turnover frequencies with a low overpotential requirement, and high selectivity for H 2 in the face of other gases. 1 A number of applications of hydrogenases have been explored, including light-driven H 2 production, 2 H 2 fuel cells, 3 and H 2 -supported recycling of the cofactor NADH. 4,5 Hydrogenases capable of functioning in the presence of O 2 are of particular interest for these applications. This impressive catalytic ability, together with the fact that hydrogenase active sites are built from readily available metals (iron, nickel), has inspired studies directed towards a detailed understanding of their chemistry. The presence of intrinsic CO and CN À ligands at the active sites means that infrared (IR) spectroscopy is a useful method for following electronic and coordination changes during catalysis or inhibition brought about by light triggers, gas exchange or electrochemical control. 6-9 Solution IR spectroelectrochemical studies interpreted alongside EPR analysis 10 and X-ray crystallographic structures have shaped our understanding of hydrogenase structure and mechanism, distinguishing a range of catalytically active and inactive states of the active site, with subtle variations evident between hydrogenases from different organisms. Most of the IR spectroelectrochemical studies on hydrogenases have been carried out in transmission geometry in an optically transparent thin layer electrochemical (OTTLE) cell originally developed by Moss et al. using a gold minigrid working electrode. 11 It is usually necessary to include small molecule redox mediators to achieve reasonable rates of electron transfer between the electrode and the hydrogenase; an exception is a study of the unusually small 49 kDa [FeFe]-hydrogenase from Chlamydomonas reinhardtii. 12 An Attenuated Total Reflectance (ATR) geometry opens up more flexible options for choice of working electrode. Rich and co-workers have developed an ATR-IR spectroelectrochemical cell in which a layer of protein coated onto the optical element is in contact with redox which shuttle electrons across the solution interface from a platinum mesh or glassy carbon electrode. 13 An alternative approach used widely for studying metallic surface chemistry, in which a thin metallic layer deposited on an ATR prism serves as the working electrode, has been adapted for biological molecules. 8,14 In this configuration, the protein is immobilised onto the gold electrode layer via a self-assembled alkanethiolate monolayer, and is in efficient solute contact, as demonstrated for reduction of hydrogenases upon introduction of H 2 . 8 Graphitic elect...

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Reduction of Unusual Iron-Sulfur Clusters in the H2-sensing Regulatory Ni-Fe Hydrogenase from Ralstonia eutropha H16

Cited by 47 publications

References 47 publications

How oxygen attacks [FeFe] hydrogenases from photosynthetic organisms

How oxygen attacks [FeFe] hydrogenases from photosynthetic organisms

Spectroscopic Insights into the Oxygen-tolerant Membrane-associated [NiFe] Hydrogenase of Ralstonia eutropha H16

Attenuated total reflectance infrared spectroelectrochemistry at a carbon particle electrode; unmediated redox control of a [NiFe]-hydrogenase solution

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