The active site of the [FeFe]-hydrogenase from Desulfovibrio desulfuricans. II. Redox properties, light sensitivity and CO-ligand exchange as observed by infrared spectroscopy

Abstract

In [FeFe]-hydrogenases, the H cluster (hydrogen-activating cluster) contains a di-iron centre ([2Fe]H subcluster, a (L)(CO)(CN)Fe(mu-RS2)(mu-CO)Fe(CysS)(CO)(CN) group) covalently attached to a cubane iron-sulphur cluster ([4Fe-4S]H subcluster). The Cys-thiol functions as the link between one iron (called Fe1) of the [2Fe]H subcluster and one iron of the cubane subcluster. The other iron in the [2Fe]H subcluster is called Fe2. The light sensitivity of the Desulfovibrio desulfuricans enzyme in a variety of states has been studied with infrared (IR) spectroscopy. The aerobic inactive enzyme (H(inact) state) and the CO-inhibited active form (H(ox)-CO state) were stable in light. Illumination of the H(ox) state led to a kind of cannibalization; in some enzyme molecules the H cluster was destroyed and the released CO was captured by the H clusters in other molecules to form the light-stable H(ox)-CO state. Illumination of active enzyme under 13CO resulted in the complete exchange of the two intrinsic COs bound to Fe2. At cryogenic temperatures, light induced the photodissociation of the extrinsic CO and the bridging CO of the enzyme in the H(ox)-CO state. Electrochemical redox titrations showed that the enzyme in the H(inact) state converts to the transition state (H(trans)) in a reversible one-electron redox step (E (m, pH 7) = -75 mV). IR spectra demonstrate that the added redox equivalent not only affects the [4Fe-4S]H subcluster, but also the di-iron centre. Enzyme in the H(trans) state reacts with extrinsic CO, which binds to Fe2. The H(trans) state converts irreversibly into the H(ox) state in a redox-dependent reaction most likely involving two electrons (E (m, pH 7) = -261 mV). These electrons do not end up on any of the six Fe atoms of the H cluster; the possible destiny of the two redox equivalents is discussed. An additional reversible one-electron redox reaction leads to the H(red) state (E (m, pH 7) = -354 mV), where both Fe atoms of the [2Fe]H subcluster have the same formal oxidation state. The possible oxidation states of Fe1 and Fe2 in the various enzyme states are discussed. Low redox potentials (below -500 mV) lead to destruction of the [2Fe]H subcluster.

Targeting Intermediates of [FeFe]-Hydrogenase by CO and CN Vibrational Signatures

“…Another complication arises from the large number of ν(CO) bands observed in the IR spectra of H red , strongly suggesting that the investigated samples contain different forms of the enzyme active site. Furthermore, the IR spectrum of H red from Chlamydomonas reinhardtii green alga reported very recently by Lubitz et al [75] differs significantly from those previously reported [62,73], and it is compatible with a CO ligand fully bridged between the two iron ions.…”

“…In view of the above mentioned experimental data on H red , it is reasonable to compare the IR frequencies of the CO-bridged isomers with the experimental spectrum reported by Lubitz et al (hereafter the corresponding form will be labelled H red (b)) [75], and the IR frequencies of the all-terminal CO isomers with the spectra reported in Ref. [73], and Ref. [62] (hereafter the corresponding form will be labelled H red (t)).…”

“…[73] and Ref. [62], as well as simulated spectra of selected bimetallic models of H red (t) are shown in Figure 8.…”

How oxygen attacks [FeFe] hydrogenases from photosynthetic organisms

“…In this article, all potential values E have been adjusted to the standard hydrogen electrode (SHE) scale by using the relationship E SHE ϭ ESCE ϩ 242 mV. The cell was blacked out with black electrical tape to avoid light-activated processes convoluting the results (7,16) and thermostated by a water-jacket linked to a water-circulator. To prepare an enzyme film, the PGE electrode was first polished for 10 s with an aqueous slurry of ␣-alumina (1 m, Buehler) and sonicated for 5 s in purified water, before 1.5 L enzyme solution (containing 0.02-0.2 g CrHydA1, i.e., up to Ϸ2 M, pH 8) was applied and removed after a few minutes.…”

“…The distal Fe is also coordinated by one CO and one CN Ϫ ligand, and an additional binding site is vacant (9) or occupied by an exchangeable O ligand, most likely a water molecule (11). In the structure of the [FeFe] hydrogenase from Desulfovibrio desulfuricans, which is believed to be crystallized in the H red form, the bridging CO is replaced by a terminal CO on Fe d (13 (16)(17)(18). Carbon monoxide is a -acceptor ligand and binds to electron-rich transition metals (19).…”