Maria-Eirini Pandelia

Membrane-bound hydrogenase I from the hyperthermophilic bacterium Aquifex aeolicus: enzyme activation, redox intermediates and oxygen tolerance.

The membrane-bound hydrogenase (Hase I) of the hyperthermophilic bacterium Aquifex aeolicus belongs to an intriguing class of redox enzymes that show enhanced thermostability and oxygen tolerance. Protein film electrochemistry is employed here to portray the interaction of Hase I with molecular oxygen and obtain an overall picture of the catalytic activity. Fourier transform infrared (FTIR) spectroscopy integrated with in situ electrochemistry is used to identify structural details of the [NiFe] site and the intermediate states involved in its redox chemistry. We found that the active site coordination is similar to that of standard hydrogenases, with a conserved Fe(CN)(2)CO moiety. However, only four catalytic intermediates could be detected; these correspond structurally to the Ni-B, Ni-SI(a), Ni-C, and Ni-R states of standard hydrogenases. The Ni-SI/Ni-C and Ni-C/Ni-R midpoint potentials are approximately 100 mV more positive than those observed in mesophilic hydrogenases, which may be the reason that A. aeolicus Hase I is more suitable as a catalyst for H(2) oxidation than production. Protein film electrochemistry shows that oxygen inhibits the enzyme by reacting at the active site to form a single species (Ni-B); the same inactive state is obtained under oxidizing, anaerobic conditions. The mechanism of anaerobic inactivation and reactivation in A. aeolicus Hase I is similar to that in standard hydrogenases. However, the reactivation of the former is more than 2 orders of magnitude faster despite the fact that reduction of Ni-B is not thermodynamically more favorable. A scheme for the enzymatic mechanism of A. aeolicus Hase I is presented, and the results are discussed in relation to the proposed models of oxygen tolerance.

Characterization of a unique [FeS] cluster in the electron transfer chain of the oxygen tolerant [NiFe] hydrogenase from Aquifex aeolicus

Iron-sulfur clusters are versatile electron transfer cofactors, ubiquitous in metalloenzymes such as hydrogenases. In the oxygen-tolerant Hydrogenase I from Aquifex aeolicus such electron “wires” form a relay to a diheme cytb, an integral part of a respiration pathway for the reduction of O2 to water. Amino acid sequence comparison with oxygen-sensitive hydrogenases showed conserved binding motifs for three iron-sulfur clusters, the nature and properties of which were unknown so far. Electron paramagnetic resonance spectra exhibited complex signals that disclose interesting features and spin-coupling patterns; by redox titrations three iron-sulfur clusters were identified in their usual redox states, a [3Fe4S] and two [4Fe4S], but also a unique high-potential (HP) state was found. On the basis of 57Fe Mössbauer spectroscopy we attribute this HP form to a superoxidized state of the [4Fe4S] center proximal to the [NiFe] site. The unique environment of this cluster, characterized by a surplus cysteine coordination, is able to tune the redox potentials and make it compliant with the [4Fe4S]3+ state. It is actually the first example of a biological [4Fe4S] center that physiologically switches between 3+, 2+, and 1+ oxidation states within a very small potential range. We suggest that the (1 + /2+) redox couple serves the classical electron transfer reaction, whereas the superoxidation step is associated with a redox switch against oxidative stress.

Inhibition of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F by carbon monoxide: An FTIR and EPR spectroscopic study

X-ray crystallographic studies [Ogata et al., J. Am. Chem. Soc. 124 (2002) 11628-11635] have shown that carbon monoxide binds to the nickel ion at the active site of the [NiFe] hydrogenase from Desulfovibriovulgaris Miyazaki F and inhibits its catalytic function. In the present work spectroscopic aspects of the CO inhibition for this bacterial organism are reported for the first time and enable a direct comparison with the existing crystallographic data. The binding affinity of each specific redox state for CO is probed by FTIR spectro-electrochemistry. It is shown that only the physiological state Ni-SI(a) reacts with CO. The CO-inhibited product state is EPR-silent (Ni2+) and exists in two forms, Ni-SCO and Ni-SCO(red). At very negative potentials, the exogenous CO is electrochemically detached from the active site and the active Ni-R states are obtained. At temperatures below 100 K, photodissociation of the extrinsic CO from the Ni-SCO state results in Ni-SI(a) that is identified to be the only light-induced state. In the dark, rebinding of CO takes place; the recombination rate constants are of biexponential character and the activation barrier is determined to be approximately 9 kJ mol(-1). In addition, formation of a paramagnetic CO-inhibited state (Ni-CO) was observed that results from the interaction of carbon monoxide with the Ni-L state. It is proposed that the nickel in Ni-CO is in a formal monovalent state (Ni1+).

A Metal–Metal Bond in the Light-Induced State of [NiFe] Hydrogenases with Relevance to Hydrogen Evolution

The light-induced Ni-L state of [NiFe] hydrogenases is well suited to investigate the identity of the amino acid base that functions as a proton acceptor in the hydrogen turnover cycle in this important class of enzymes. Density functional theory calculations have been performed on large models that include the complete [NiFe] center and parts of the second coordination sphere. Combined with experimental data, in particular from electron paramagnetic resonance and Fourier transform infrared (FTIR) spectroscopy, the calculations indicate that the hydride ion, which is located in the bridging position between nickel and iron in the Ni-C state, dissociates upon illumination as a proton and binds to a nearby thiolate base. Moreover, the formation of a functionally relevant nickel-iron bond upon dissociation of the hydride is unequivocally observed and is in full agreement with the observed g values, ligand hyperfine coupling constants, and FTIR stretching frequencies. This metal-metal bond can be protonated and thus functions like a base. The nickel-iron bond is important for all intermediates with an empty bridge in the catalytic cycle, and the electron pair that constitutes this bond thus plays a crucial role in the hydrogen evolution catalyzed by the enzyme.

Electronic structure of the unique [4Fe-3S] cluster in O2-tolerant hydrogenases characterized by 57Fe Mössbauer and EPR spectroscopy

Iron–sulfur clusters are ubiquitous electron transfer cofactors in hydrogenases. Their types and redox properties are important for H2 catalysis, but, recently, their role in a protection mechanism against oxidative inactivation has also been recognized for a [4Fe-3S] cluster in O2-tolerant group 1 [NiFe] hydrogenases. This cluster, which is uniquely coordinated by six cysteines, is situated in the proximity of the catalytic [NiFe] site and exhibits unusual redox versatility. The [4Fe-3S] cluster in hydrogenase (Hase) I from Aquifex aeolicus performs two redox transitions within a very small potential range, forming a superoxidized state above +200 mV vs. standard hydrogen electrode (SHE). Crystallographic data has revealed that this state is stabilized by the coordination of one of the iron atoms to a backbone nitrogen. Thus, the proximal [4Fe-3S] cluster undergoes redox-dependent changes to serve multiple purposes beyond classical electron transfer. In this paper, we present field-dependent 57Fe-Mössbauer and EPR data for Hase I, which, in conjunction with spectroscopically calibrated density functional theory (DFT) calculations, reveal the distribution of Fe valences and spin-coupling schemes for the iron–sulfur clusters. The data demonstrate that the electronic structure of the [4Fe-3S] core in its three oxidation states closely resembles that of corresponding conventional [4Fe-4S] cubanes, albeit with distinct differences for some individual iron sites. The medial and distal iron–sulfur clusters have similar electronic properties as the corresponding cofactors in standard hydrogenases, although their redox potentials are higher.

The oxygen-tolerant hydrogenase I from Aquifex aeolicus weakly interacts with carbon monoxide: an electrochemical and time-resolved FTIR study.

The [NiFe] hydrogenase (Hase I) involved in the aerobic respiration of the hyperthermophilic bacterium Aquifex aeolicus shows increased oxygen tolerance and thermostability and can form very stable films on pyrolytic graphite electrodes. Oxygen-tolerant enzymes, like the ones from A. aeolicus and Ralstonia eutropha, are reported to be insensitive to CO inhibition. This is in contrast to known and well-characterized (oxygen-sensitive) hydrogenases, for which carbon monoxide is a competitive inhibitor. In this study, the interaction of Hase I from A. aeolicus with CO is examined using in situ infrared electrochemistry and time-resolved FTIR spectroscopy. We could observe the formation of a CO adduct state, a finding that set the grounds to investigate the affinity of an O(2)-tolerant enzyme for binding CO as well as the reversibility of this process. In the case of A. aeolicus, this extrinsic CO is shown to be weakly attached and the adduct state is light-sensitive at low temperatures. The energetic parameters for the rebinding of CO at the active site were estimated from the rate constants of this process after photolysis and the results compared to those obtained for standard hydrogenases. Formation of a weak Ni-CO bond in the active site of Hase I most likely results from the different interaction of this enzyme with inhibitors and/or different active site electronic properties to which non standard amino acid residues in the vicinity of the active site might contribute.

Human anamorsin binds [2Fe–2S] clusters with unique electronic properties

The eukaryotic anamorsin protein family, which has recently been proposed to be part of an electron transfer chain functioning in the early steps of cytosolic iron–sulfur (Fe/S) protein biogenesis, is characterized by a largely unstructured domain (CIAPIN1) containing two conserved cysteine-rich motifs (CX8CX2CXC and CX2CX7CX2C) whose Fe/S binding properties and electronic structures are not well defined. Here, we found that (1) each motif in human anamorsin is able to bind independently a [2Fe–2S] cluster through its four cysteine residues, the binding of one cluster mutually excluding the binding of the second, (2) the reduced [2Fe–2S]+ clusters exhibit a unique electronic structure with considerable anisotropy in their coordination environment, different from that observed in reduced, plant-type and vertebrate-type [2Fe–2S] ferredoxin centers, (3) the reduced cluster bound to the CX2CX7CX2C motif reveals an unprecedented valence localization-to-delocalization transition as a function of temperature, and (4) only the [2Fe–2S] cluster bound to the CX8CX2CXC motif is involved in the electron transfer with its physiological protein partner Ndor1. The unique electronic properties of both [2Fe–2S] centers can be interpreted by considering that both cysteine-rich motifs are located in a highly unstructured and flexible protein region, whose local conformational heterogeneity can induce anisotropy in metal coordination. This study contributes to the understanding of the functional role of the CIAPIN1 domain in the anamorsin family, suggesting that only the [2Fe–2S] cluster bound to the CX8CX2CXC motif is indispensable in the electron transfer chain assembling cytosolic Fe/S proteins.Graphical abstract

Electronic Structure of the Quinone Radical Anion A1˙― of Photosystem I Investigated by Advanced Pulse EPR and ENDOR Techniques

Vitamin K1 (VK1) is an important cofactor of the electron-transfer chain in photosystem I (PS I), referred to as A1. The special properties of this quinone result from its unique interaction(s) with the protein surrounding. In particular, a single H-bond to neutral A1 was identified previously in the X-ray crystal structure of PS I. During light-induced electron transfer in PS I, A1 is transiently reduced to the radical anion A1*-. In this work, we characterized the electron spin density distribution of A1*- with the aim of understanding the influence of the protein surrounding on it. We studied the light-induced spin-polarized radical pair P700*+A1*- and the photoaccumulated radical anion A1*-, using advanced pulse EPR, ENDOR, and TRIPLE techniques at Q-band (34 GHz). Exchange with fully deuterated quinone in the A1 binding site allowed differentiation between proton hyperfine couplings from the quinone and from the protein surrounding. In addition, DFT calculations on a model of the A1 site were performed and provided proton hyperfine couplings that were in close agreement with the ones determined experimentally. This combined approach allowed the assignment of proton hyperfine coupling tensors to molecular positions, thereby yielding a picture of the spin density distribution in A1*-. Comparison with VK1*- in organic solvents (Epel et al. J. Phys. Chem. B 2006, 110, 11549.) leads to the conclusion that the single H-bond present in both the radical pair P700*+A1*- and the photoaccumulated radical anion A1*- is, indeed, the crucial factor that governs the electronic structure of A1*-.

Evidence for a catalytically and kinetically competent enzyme-substrate cross-linked intermediate in catalysis by lipoyl synthase.

Lipoyl synthase (LS) catalyzes the final step in lipoyl cofactor biosynthesis: the insertion of two sulfur atoms at C6 and C8 of an (N6-octanoyl)-lysyl residue on a lipoyl carrier protein (LCP). LS is a member of the radical SAM superfamily, enzymes that use a [4Fe–4S] cluster to effect the reductive cleavage of S-adenosyl-l-methionine (SAM) to l-methionine and a 5′-deoxyadenosyl 5′-radical (5′-dA•). In the LS reaction, two equivalents of 5′-dA• are generated sequentially to abstract hydrogen atoms from C6 and C8 of the appended octanoyl group, initiating sulfur insertion at these positions. The second [4Fe–4S] cluster on LS, termed the auxiliary cluster, is proposed to be the source of the inserted sulfur atoms. Herein, we provide evidence for the formation of a covalent cross-link between LS and an LCP or synthetic peptide substrate in reactions in which insertion of the second sulfur atom is slowed significantly by deuterium substitution at C8 or by inclusion of limiting concentrations of SAM. The observation that the proteins elute simultaneously by anion-exchange chromatography but are separated by aerobic SDS-PAGE is consistent with their linkage through the auxiliary cluster that is sacrificed during turnover. Generation of the cross-linked species with a small, unlabeled (N6-octanoyl)-lysyl-containing peptide substrate allowed demonstration of both its chemical and kinetic competence, providing strong evidence that it is an intermediate in the LS reaction. Mössbauer spectroscopy of the cross-linked intermediate reveals that one of the [4Fe–4S] clusters, presumably the auxiliary cluster, is partially disassembled to a 3Fe-cluster with spectroscopic properties similar to those of reduced [3Fe–4S]0 clusters.

FTIR study on the light sensitivity of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F: Ni-C to Ni-L photoconversion, kinetics of proton rebinding and H/D isotope effect.

The light-induced Ni-C to Ni-L transition results in the dissociation of a hydrogenic species, originating from the dihydrogen splitting at the active site. Back conversion in the dark to form Ni-C was investigated by studying the rebinding kinetics of this ligand in protonated (H(2)/H(2)O) and deuterated (D(2)/D(2)O) samples using time resolved FTIR spectroscopy.