Direct Detection of a Hydrogen Ligand in the [NiFe] Center of the Regulatory H₂-Sensing Hydrogenase fromRalstoniaeutrophain Its Reduced State by HYSCORE and ENDOR Spectroscopy

Abstract

The regulatory H2-sensing [NiFe] hydrogenase of the beta-proteobacterium Ralstonia eutropha displays an Ni-C "active" state after reduction with H2 that is very similar to the reduced Ni-C state of standard [NiFe] hydrogenases. Pulse electron nuclear double resonance (ENDOR) and four-pulse ESEEM (hyperfine sublevel correlation, HYSCORE) spectroscopy are applied to obtain structural information on this state via detection of the electron-nuclear hyperfine coupling constants. Two proton hyperfine couplings are determined by analysis of ENDOR spectra recorded over the full magnetic field range of the EPR spectrum. These are associated with nonexchangeable protons and belong to the beta-CH(2) protons of a bridging cysteine of the NiFe center. The signals of a third proton exhibit a large anisotropic coupling (Ax = 18.4 MHz, Ay = -10.8 MHz, Az = -18 MHz). They disappear from the 1H region of the ENDOR spectra after exchange of H2O with 2H2O and activation with 2H2 instead of H2 gas. They reappear in the 2H region of the ENDOR and HYSCORE spectra. Based on a comparison with the spectroscopically similar [NiFe] hydrogenase of Desulfovibrio vulgaris Miyazaki F, for which the g-tensor orientation of the Ni-C state with respect to the crystal structure is known (Foerster et al. J. Am. Chem. Soc. 2003, 125, 83-93), an assignment of the 1H hyperfine couplings is proposed. The exchangeable proton resides in a bridging position between the Ni and Fe and is assigned to a formal hydride ion. After illumination at low temperature (T = 10 K), the Ni-L state is formed. For the Ni-L state, the strong hyperfine coupling observed for the exchangeable hydrogen in Ni-C is lost, indicating a cleavage of the metal-hydride bond(s). These experiments give first direct information on the position of hydrogen binding in the active NiFe center of the regulatory hydrogenase. It is proposed that such a binding situation is also present in the active Ni-C state of standard hydrogenases.

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

“…H 2 -mediated reductive activation of the Ni u -A state is a long term process and requires hours until the oxygen species is completely removed from the active site. In contrast, only seconds of incubation with H 2 are required to convert the Ni r -B state into the catalytically active, EPR-detectable Ni a -C state in which a hydride occupies the bridging position between nickel and iron (19,20). An overview of the different redox states of the [NiFe] active site is shown in Fig.…”

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

“…This state does not need to be activated but is always ready to bind hydrogen, a prerequisite for the sensor function (10,11). In the presence of H 2 it is rapidly converted to a state revealing a typical EPR-signal, termed Ni-C, due to a Ni(III)-H Ϫ species formed during heterolytic H 2 cleavage (15). In standard Ni-Fe hydrogenases the nickel is coordinated by four conserved cysteine residues.…”

“…Although information has become available about the sequence of events that occur at the Ni-Fe active site upon interaction of the RH with H 2 (10,11,15), it is unclear whether electron transfer out of the Ni-Fe site takes place during H 2 cleavage and to where these electrons are transferred. Information on these points is expected to contribute to the understanding of the H 2 -sensing mechanism of the RH-HoxJ complex (14).…”

“…Therefore, it was postulated that it might also harbor three Fe-S clusters (6). EPR investigation of the RH, however, did not show reduced Fe-S clusters when the Ni-C EPR signal was formed under H 2 (3,10,15). It has been proposed that a non-Fe-S cofactor may be involved in electron transfer instead (10).…”

Characterization of the FeH Bond in a Three‐Coordinate Terminal Hydride Complex of Iron(I)

“…[7][8][9][10][11] As these biological hydrides arise in catalytic intermediates that are not amenable to crystallographic characterization, it is essential to identify the spectroscopic signatures of crystallographically characterized transition-metal hydride complexes. [12] As ENDOR spectroscopy of odd-electron enzyme intermediates has been a central tool in characterizing biological iron hydrides, [7][8][9][13][14][15] a key advance would be the preparation of simple mononuclear Fe-H complexes with an odd-electron iron(I) or iron(III) oxidation state for comparison. However, crystallographically characterized compounds of this type have been unknown, presumably because of the low oxidation state and the high reactivity of hydrides.…”