Ingo Zebger

A unique iron-sulfur cluster is crucial for oxygen tolerance of a [NiFe]-hydrogenase

Hydrogenases are essential for H(2) cycling in microbial metabolism and serve as valuable blueprints for H(2)-based biotechnological applications. However, most hydrogenases are extremely oxygen sensitive and prone to inactivation by even traces of O(2). The O(2)-tolerant membrane-bound [NiFe]-hydrogenase of Ralstonia eutropha H16 is one of the few examples that can perform H(2) uptake in the presence of ambient O(2). Here we show that O(2) tolerance is crucially related to a modification of the internal electron-transfer chain. The iron-sulfur cluster proximal to the active site is surrounded by six instead of four conserved coordinating cysteines. Removal of the two additional cysteines alters the electronic structure of the proximal iron-sulfur cluster and renders the catalytic activity sensitive to O(2) as shown by physiological, biochemical, spectroscopic and electrochemical studies. The data indicate that the mechanism of O(2) tolerance relies on the reductive removal of oxygenic species guided by the unique architecture of the electron relay rather than a restricted access of O(2) to the active site.

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

This study provides the first spectroscopic characterization of the membrane-bound oxygen-tolerant [NiFe] hydrogenase (MBH) from Ralstonia eutropha H16 in its natural environment, the cytoplasmic membrane. The H2-converting MBH is composed of a large subunit, harboring the [NiFe] active site, and a small subunit, capable in coordinating one [3Fe4S] and two [4Fe4S] clusters. The hydrogenase dimer is electronically connected to a membrane-integral cytochrome b. EPR and Fourier transform infrared spectroscopy revealed a strong similarity of the MBH active site with known [NiFe] centers from strictly anaerobic hydrogenases. Most redox states characteristic for anaerobic [NiFe] hydrogenases were identified except for one remarkable difference. The formation of the oxygen-inhibited Niu-A state was never observed. Furthermore, EPR data showed the presence of an additional paramagnetic center at high redox potential (+290 mV), which couples magnetically to the [3Fe4S] center and indicates a structural and/or redox modification at or near the proximal [4Fe4S] cluster. Additionally, significant differences regarding the magnetic coupling between the Nia-C state and [4Fe4S] clusters were observed in the reduced form of the MBH. The spectroscopic properties are discussed with regard to the unusual oxygen tolerance of this hydrogenase and in comparison with those of the solubilized, dimeric form of the MBH.

Overexpression, Isolation, and Spectroscopic Characterization of the Bidirectional [NiFe] Hydrogenase from Synechocystis sp. PCC 6803

The bidirectional [NiFe] hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 was purified to apparent homogeneity by a single affinity chromatography step using a Synechocystis mutant with a Strep-tag II fused to the C terminus of HoxF. To increase the yield of purified enzyme and to test its overexpression capacity in Synechocystis the psbAII promoter was inserted upstream of the hoxE gene. In addition, the accessory genes (hypF, C, D, E, A, and B) from Nostoc sp. PCC 7120 were expressed under control of the psbAII promoter. The respective strains show higher hydrogenase activities compared with the wild type. For the first time a Fourier transform infrared (FTIR) spectroscopic characterization of a [NiFe] hydrogenase from an oxygenic phototroph is presented, revealing that two cyanides and one carbon monoxide coordinate the iron of the active site. At least four different redox states of the active site were detected during the reversible activation/inactivation. Although these states appear similar to those observed in standard [NiFe] hydrogenases, no paramagnetic nickel state could be detected in the fully oxidized and reduced forms. Electron paramagnetic resonance spectroscopy confirms the presence of several iron-sulfur clusters after reductive activation. One [4Fe4S]+ and at least one [2Fe2S]+ cluster could be identified. Catalytic amounts of NADH or NADPH are sufficient to activate the reaction of this enzyme with hydrogen.

Probing the active site of an O2-tolerant NAD+-reducing [NiFe]-hydrogenase from Ralstonia eutropha H16 by in situ EPR and FTIR spectroscopy.

[NiFe]-hydrogenases catalyze the reversible cleavage of dihydrogen into two protons and two electrons. This process plays an important role in the energy metabolism of many microorganisms. For most [NiFe]-hydrogenases, the process of H2 cycling is extremely sensitive to molecular oxygen as O2 exhibits a high affinity to the active site. However, some organisms are capable of catalyzing H2 cycling even at ambient oxygen levels. Notably, the b-proteobacterium Ralstonia eutropha H16 (Re) harbors three different [NiFe]hydrogenases, all of which display a remarkable oxygentolerance. The underlying molecular mechanisms are not yet fully understood. For the regulatory hydrogenase (RH) of Re, a narrow gas tunnel is thought to restrict O2 access to the active site. The Re membrane-bound hydrogenase (MBH) has a high redox potential FeS cluster in close proximity to the active site, a property that might be related to the observation that O2-inhibited MBH re-activates rapidly at high potentials. The soluble hydrogenase (SH) of Re is a cytoplasmic NAD-reducing six-subunit enzyme that is closely related to cyanobacterial bidirectional [NiFe]-hydrogenases. 7] For purified SH, a modified catalytic site was proposed on the basis of numerous biochemical and spectroscopic studies. 8, 9] In contrast to “standard” [NiFe]-hydrogenases, in which the active site iron is kept in the low-spin iron(II) state by one carbonyl and two cyanide ligands, Fourier transform infrared (FTIR) spectroscopy and concomitant chemical analysis suggested one additional cyanide bound to each metal ion of the catalytic center. The nickelbound cyanide ligand has been proposed to prevent the formation of the so-called Niu-A state, which is the most oxidized, O2-inactivated state in [NiFe]-hydrogenases. [9]

Surface-enhanced vibrational spectroscopy for probing transient interactions of proteins with biomimetic interfaces: electric field effects on structure, dynamics and function of cytochrome c

Most of the biochemical and biophysical processes of proteins take place at membranes, and are thus under the influence of strong local electric fields, which are likely to affect the structure as well as the reaction mechanism and dynamics. To analyse such electric field effects, biomimetic interfaces may be employed that consist of membrane models deposited on nanostructured metal electrodes. For such devices, surface‐enhanced resonance Raman and IR absorption spectroscopy are powerful techniques to disentangle the complex interfacial processes of proteins in terms of rotational diffusion, electron transfer, and protein and cofactor structural changes. The present article reviews the results obtained for the haem protein cytochrome c, which is widely used as a model protein for studying the various reaction steps of interfacial redox processes in general. In addition, it is shown that electric field effects may be functional for the natural redox processes of cytochrome c in the respiratory chain, as well as for the switch from the redox to the peroxidase function, one of the key events preceding apoptosis.

Carbamoylphosphate serves as the source of CN−, but not of the intrinsic CO in the active site of the regulatory [NiFe]-hydrogenase from Ralstonia eutropha

Within the catalytic centre of [NiFe]‐hydrogenases one carbonyl and two cyanide ligands are covalently attached to the iron. To identify the metabolic origins of these ligands, the regulatory [NiFe] hydrogenase in conjunction with the indigenous Hyp maturation proteins of Ralstonia eutropha H16 were heterologously overproduced in E. coli grown in the presence of l‐[ureido‐13C] citrulline and NaH13CO3. Infrared spectroscopy of purified hydrogenase provided direct evidence that only the cyanide ligands, but not the CO ligand, originate from CO2 and carbamoylphosphate. Incorporation of label from 13CO exclusively into the carbonyl ligand indicates that free CO is a possible precursor in carbonyl ligand biosynthesis.

Direct Optical Detection of Singlet Oxygen from a Single Cell

Abstract Singlet oxygen has been detected in single nerve cells by its weak 1270 nm phosphorescence (a1Δg→X3Σg−) upon irradiation of a photosensitizer incorporated in the cell. Thus, one can now consider the application of direct optical imaging techniques to mechanistic studies of singlet oxygen at the single-cell level.

Reversible [4Fe-3S] cluster morphing in an O2-tolerant [NiFe] hydrogenase

Hydrogenases catalyze the reversible oxidation of H(2) into protons and electrons and are usually readily inactivated by O(2). However, a subgroup of the [NiFe] hydrogenases, including the membrane-bound [NiFe] hydrogenase from Ralstonia eutropha, has evolved remarkable tolerance toward O(2) that enables their host organisms to utilize H(2) as an energy source at high O(2). This feature is crucially based on a unique six cysteine-coordinated [4Fe-3S] cluster located close to the catalytic center, whose properties were investigated in this study using a multidisciplinary approach. The [4Fe-3S] cluster undergoes redox-dependent reversible transformations, namely iron swapping between a sulfide and a peptide amide N. Moreover, our investigations unraveled the redox-dependent and reversible occurence of an oxygen ligand located at a different iron. This ligand is hydrogen bonded to a conserved histidine that is essential for H(2) oxidation at high O(2). We propose that these transformations, reminiscent of those of the P-cluster of nitrogenase, enable the consecutive transfer of two electrons within a physiological potential range.

Concerted Action of Two Novel Auxiliary Proteins in Assembly of the Active Site in a Membrane-bound [NiFe] Hydrogenase

[NiFe] hydrogenases catalyze the reversible conversion of H2 into protons and electrons. The reaction takes place at the active site, which is composed of a nickel and an iron atom and three diatomic ligands, two cyanides and one carbon monoxide, bound to the iron. The NiFe(CN-)2CO cofactor is synthesized by an intricate posttranslational maturation process, which is mediated by a set of six conserved Hyp proteins. Depending on the cellular location and the physiological function, additional auxiliary proteins are involved in hydrogenase biosynthesis. Here we present evidence that the auxiliary proteins HoxL and HoxV assist in assembly of the Fe(CN-)2CO moiety. This unit was identified as a cofactor intermediate of the oxygen-tolerant membrane-bound [NiFe] hydrogenase (MBH) in the β-proteobacterium Ralstonia eutropha H16. Both HoxL and HoxV proved to be essential for H2-oxidizing activity and MBH-driven growth on H2. Copurification studies revealed that HoxL and HoxV directly interact with the hydrogenase apoprotein. HoxV forms complexes with HoxL and HypC, a HoxL paralogue that is essential for cofactor assembly. These observations suggest that HoxL acts as a specific chaperone assisting the transfer of the Fe(CN-)2CO cofactor intermediate from the Hyp machinery to the MBH. This shuttle also involves the scaffold protein HoxV. Indeed, infrared spectroscopy and metal analysis identified for the first time a non-redox-active Fe(CN-)2CO intermediate coordinated to HoxV.

NAD(H)-coupled hydrogen cycling – structure–function relationships of bidirectional [NiFe] hydrogenases

Hydrogenases catalyze the activation or production of molecular hydrogen. Due to their potential importance for future biotechnological applications, these enzymes have been in the focus of intense research for the past decades. Bidirectional [NiFe] hydrogenases are of particular interest as they couple the reversible cleavage of hydrogen to the redox conversion of NAD(H). In this account, we review the current state of knowledge about mechanistic aspects and structural determinants of these complex multi‐cofactor enzymes. Special emphasis is laid on the oxygen‐tolerant NAD(H)‐linked bidirectional [NiFe] hydrogenase from Ralstonia eutropha.