Eduard Reijerse

Biomimetic assembly and activation of [FeFe]-hydrogenases

Hydrogenases are the most active molecular catalysts for hydrogen production and uptake, and could therefore facilitate the development of new types of fuel cell. In [FeFe]-hydrogenases, catalysis takes place at a unique di-iron centre (the [2Fe] subsite), which contains a bridging dithiolate ligand, three CO ligands and two CN– ligands. Through a complex multienzymatic biosynthetic process, this [2Fe] subsite is first assembled on a maturation enzyme, HydF, and then delivered to the apo-hydrogenase for activation. Synthetic chemistry has been used to prepare remarkably similar mimics of that subsite, but it has failed to reproduce the natural enzymatic activities thus far. Here we show that three synthetic mimics (containing different bridging dithiolate ligands) can be loaded onto bacterial Thermotoga maritima HydF and then transferred to apo-HydA1, one of the hydrogenases of Chlamydomonas reinhardtii algae. Full activation of HydA1 was achieved only when using the HydF hybrid protein containing the mimic with an azadithiolate bridge, confirming the presence of this ligand in the active site of native [FeFe]-hydrogenases. This is an example of controlled metalloenzyme activation using the combination of a specific protein scaffold and active-site synthetic analogues. This simple methodology provides both new mechanistic and structural insight into hydrogenase maturation and a unique tool for producing recombinant wild-type and variant [FeFe]-hydrogenases, with no requirement for the complete maturation machinery.

Multi-frequency EPR spectroscopy of myoglobin. Spectral effects for high-spin iron(III) ion at high magnetic fields

We report the result of a series of EPR experiments performed at microwave frequencies from 1 to 285 GHz on the S = 5/2 iron(III) ion in acid metmyoglobin. Two important phenomena occur upon increasing the frequency. Firstly, the effective g⊥-value decreases notably as the Zeeman energy approaches the value of D, the zero-field splitting. Secondly, the linewidth of the spectra increases anomalously above 130 GHz. The dependence of observed linewidth on the microwave frequency shows three distinct phases. At low frequencies, broadening by unresolved hyperfine splittings is dominant. It is shown that from X-band frequencies onwards, a distribution in the value of g⊥ and D causes the measured dependence of linewidth on frequency. The implications of this experiment for the study of randomly distributed spin systems at high frequencies are discussed.

A CW and pulse EPR spectrometer operating at 122 and 244 GHz using a quasi-optical bridge and a cryogen-free 12 T superconducting magnet

A high-field continuous-wave (CW) and pulse electron paramagnetic resonance spectrometer operating at 122 and 244 GHz is described. The instrument is based on a millimeter-wave bridge built from quasi-optical components. To improve the sensitivity, a cryo-cooled detector/mixer is used. The magnetic field is generated using a cryogen-free superconducting 12 T magnet (warm bore, 88 mm) equipped with a helium-flow cryostat for sample cooling. The advantages of this spectrometer are described and first results (obtained in CW mode) on different types of samples at 122 and 244 GHz are presented. The extensions to pulse operation as well as double resonance techniques (electron-electron and electron-nuclear) are briefly discussed.

Empirical and computational design of iron-sulfur cluster proteins.

Here, we compare two approaches of protein design. A computational approach was used in the design of the coiled-coil iron-sulfur protein, CCIS, as a four helix bundle binding an iron-sulfur cluster within its hydrophobic core. An empirical approach was used for designing the redox-chain maquette, RCM as a four-helix bundle assembling iron-sulfur clusters within loops and one heme in the middle of its hydrophobic core. We demonstrate that both ways of design yielded the desired proteins in terms of secondary structure and cofactors assembly. Both approaches, however, still have much to improve in predicting conformational changes in the presence of bound cofactors, controlling oligomerization tendency and stabilizing the bound iron-sulfur clusters in the reduced state. Lessons from both ways of design and future directions of development are discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Spectroscopic investigations of a semi-synthetic [FeFe] hydrogenase with propane di-selenol as bridging ligand in the binuclear subsite: comparison to the wild type and propane di-thiol variants

Abstract[FeFe] Hydrogenases catalyze the reversible conversion of H2 into electrons and protons. Their catalytic site, the H-cluster, contains a generic [4Fe–4S]H cluster coupled to a [2Fe]H subsite [Fe2(ADT)(CO)3(CN)2]2−, ADT = µ(SCH2)2NH. Heterologously expressed [FeFe] hydrogenases (apo-hydrogenase) lack the [2Fe]H unit, but this can be incorporated through artificial maturation with a synthetic precursor [Fe2(ADT)(CO)4(CN)2]2−. Maturation with a [2Fe] complex in which the essential ADT amine moiety has been replaced by CH2 (PDT = propane-dithiolate) results in a low activity enzyme with structural and spectroscopic properties similar to those of the native enzyme, but with simplified redox behavior. Here, we study the effect of sulfur-to-selenium (S-to-Se) substitution in the bridging PDT ligand incorporated in the [FeFe] hydrogenase HydA1 from Chlamydomonas reinhardtii using magnetic resonance (EPR, NMR), FTIR and spectroelectrochemistry. The resulting HydA1-PDSe enzyme shows the same redox behavior as the parent HydA1-PDT. In addition, a state is observed in which extraneous CO is bound to the open coordination site of the [2Fe]H unit. This state was previously observed only in the native enzyme HydA1-ADT and not in HydA1-PDT. The spectroscopic features and redox behavior of HydA1-PDSe, resulting from maturation with [Fe2(PDSe)(CO)4(CN)2]2−, are discussed in terms of spin and charge density shifts and provide interesting insight into the electronic structure of the H-cluster. We also studied the effect of S-to-Se substitution in the [4Fe–4S] subcluster. The reduced form of HydA1 containing only the [4Fe–4Se]H cluster shows a characteristic S = 7/2 spin state which converts back into the S = 1/2 spin state upon maturation with a [2Fe]–PDT/ADT complex.

Spectroscopic Characterization of the Molybdenum Cofactor of the Sulfane Dehydrogenase SoxCD from Paracoccus pantotrophus

The bacterial sulfane dehydrogenase SoxCD is a distantly related member of the sulfite oxidase (SO) enzyme family that is proposed to oxidize protein-bound sulfide (sulfane) of SoxY as part of a multienzyme mechanism of thiosulfate metabolism. This study characterized the molybdenum cofactor of SoxCD1, comprising the catalytic molybdopterin subunit SoxC and the truncated c-type cytochrome subunit SoxD1. Electron paramagnetic resonance spectroscopy of the Mo(V) intermediate generated by dithionite reduction revealed low- and high-pH species with g and A((95,97)Mo) matrices nearly identical to those of SO, indicating a similar pentacoordinate active site in SoxCD1. However, no sulfite-induced reduction to Mo(V) was detected, nor could a strongly coupled (1)H signal or a phosphate-inhibited species be generated. This indicates that the outer coordination sphere controls substrate binding in SoxCD, permitting access only to protein-bound sulfur via the C-terminal tail of SoxY.

Chapter 11:Structure and Function of Hydrogenase Enzymes

The understanding of the basic principles of hydrogen production and utilization by the enzyme hydrogenase is a goal of major importance both for basic research and possible applications in our society. Hydrogenases are enzymes that facilitate the uptake and release of molecular hydrogen using a heterolytic reaction mechanism: H2⇌H++H−⇌2H++2e−. The acidity of H2, which is extremely low, is dramatically increased by binding to a metal. Many of the currently used catalysts for anthropogenic utilization of hydrogen involve precious metals such as platinum, while Natures catalysts are based on cheap and abundant first row transition metals. Three phylogenetically distinct classes of hydrogenase are known; these are the [NiFe], the [FeFe] and the [Fe] hydrogenases. The first two classes have active sites containing binuclear metal cores with an unusual ligand sphere, whereas the third class harbors a mononuclear iron next to a special organic cofactor. In all these hydrogenases, the protein plays an important role for tuning the active site properties, but also by providing pathways for protons, electrons as well as dihydrogen. An important feature of the native systems is the very high turnover frequency (up to ∼104 s−1). Hydrogenases from (hyper)thermophilic organisms show a remarkable stability at high temperatures (up to ∼100°C) and several [NiFe] hydrogenases (e.g. from Knallgas bacteria) are active even in the presence of ambient levels of molecular oxygen. As discussed in this chapter, a combination of X-ray crystallography, spectroscopy, electrochemistry and quantum chemistry was instrumental in characterizing the hydrogenases with respect to their structure and function. Furthermore, mechanisms for the enzymatic reactions are proposed and guidelines for the construction of biomimetic hydrogenase model systems are provided.