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.

In the catalytic cycle of [NiFe] hydrogenase the paramagnetic Ni-C intermediate is of key importance, since it is believed to carry the substrate hydrogen, albeit in a yet unknown geometry. Upon illumination at low temperatures, Ni-C is converted to the so-called Ni-L state with markedly different spectroscopic parameters. It is suspected that Ni-L has lost the "substrate hydrogen". In this work, both paramagnetic states have been generated in single crystals obtained from the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F. Evaluation of the orientation dependent spectra yielded the magnitudes of the g tensors and their orientations in the crystal axes system for both Ni-C and Ni-L. The g tensors could further be related to the atomic structure by comparison with the X-ray crystallographic structure of the reduced enzyme. Although the g tensor magnitudes of Ni-C and Ni-L are quite different, the orientations of the resulting g tensors are very similar but differ from those obtained earlier for Ni-A and Ni-B (Trofanchuk et al. J. Biol. Inorg. Chem. 2000, 5, 36-44). The g tensors were also calculated by density functional theory (DFT) methods using various structural models of the active site. The calculated g tensor of Ni-C is, concerning magnitudes and orientation, in good agreement with the experimental one for a formal Ni(III) oxidation state with a hydride (H(-)) bridge between the Ni and the Fe atom. Satisfying agreement is obtained for the Ni-L state when a formal Ni(I) oxidation state is assumed for this species with a proton (H(+)) removed from the bridge between the nickel and the iron atom.

Electron nuclear double resonance (ENDOR) and hyperfine sublevel correlation spectroscopy (HYSCORE) are applied to study the active site of catalytic [NiFe]-hydrogenase from Desulfovibrio vulgaris Miyazaki F in the reduced Ni-C state. These techniques offer a powerful tool for detecting nearby magnetic nuclei, including a metal-bound substrate hydrogen, and for mapping the spin density distribution of the unpaired electron at the active site. The observed hyperfine couplings are assigned via comparison with structural data from X-ray crystallography and knowledge of the complete g-tensor in the Ni-C state (Foerster et al. (2003) J Am Chem Soc 125:83-93). This is found to be in good agreement with density functional theory calculations. The two most strongly coupled protons (a(iso)=13.7, 11.8 MHz) are assigned to the beta-CH(2) protons of the nickel-coordinating cysteine 549, and a third proton (a(iso)=8.9 MHz) is assigned to a beta-CH(2) proton of cysteine 546. Using D(2)O exchange experiments, the presence of a hydride in the bridging position between the nickel and iron-recently been detected for a regulatory hydrogenase (Brecht et al. (2003) J Am Chem Soc 125:13075-13083)-is experimentally confirmed for the first time for catalytic hydrogenases. The hydride exhibits a small isotropic hyperfine coupling constant (a(iso)=-3.5 MHz) since it is bound to Ni in a direction perpendicular to the z-axis of the Ni (3d(z)(2)) orbital. Nitrogen signals that belong to the nitrogen N(epsilon) of His-88 have been identified. This residue forms a hydrogen bond with the spin-carrying Ni-coordinated sulfur of Cys-549. Comparison with other hydrogenases reveals that the active site is essentially the same in all proteins, including a regulatory hydrogenase.

The catalytic center of the [NiFe] hydrogenase of Desulfovibrio vulgaris Miyazaki F in the oxidized states was investigated by electron paramagnetic resonance and electron-nuclear double resonance spectroscopy applied to single crystals of the enzyme. The experimental results were compared with density functional theory (DFT) calculations. For the Ni-B state, three hyperfine tensors could be determined. Two tensors have large isotropic hyperfine coupling constants and are assigned to the beta-CH2 protons of the Cys-549 that provides one of the bridging sulfur ligands between Ni and Fe in the active center. From a comparison of the orientation of the third hyperfine tensor with the tensor obtained from DFT calculations an OH- bridging ligand has been identified in the Ni-B state. For the Ni-A state broader signals were observed. The signals of the third proton, as observed for the "ready" state Ni-B, were not observed at the same spectral position for Ni-A, confirming a structural difference involving the bridging ligand in the "unready" state of the enzyme.

The interaction between two paramagnetic metal centers, a [3Fe-4S](+) cluster and a [NiFe] center, is investigated in the hydrogenase from Desulfovibrio vulgaris Miyazaki F by pulsed ELDOR (electron-electron double resonance). The distance between the metal centers is known from X-ray crystallography. The experimental dipolar spin-spin interaction deviates from the value expected for two point-dipoles located at the centers of the metal clusters. An extended spin-coupling model accounting for the spin coupling in the [3Fe-4S](+) cluster yields the observed interaction under the assumption of a particular magnetic coupling scheme for the three Fe ions. These results demonstrate that pulsed ELDOR can be used to gain insight into the inner structure of a multinuclear metal cluster.

Single-molecule spectroscopy at cryogenic temperatures was used to elucidate spectral properties, heterogeneities, and dynamics of the chlorophyll a (Chla) molecules responsible for the fluorescence in photosystem I (PSI) from the cyanobacteria Thermosynechococcus elongatus. Absorption and hole burning data suggest the presence of three pools absorbing at wavelengths greater than 700 nm with their absorption maxima at 708, 715, and 719 nm. The responsible Chla molecules are termed C708, C715, and C719. In the emission spectra of single PSI complexes, zero-phonon lines (ZPLs) were observed over the whole red emission range of PSI. The spectral region of the C708 pool is dominated by intense ZPLs; on the other hand, the broad emission of C715/C719 is unstructured and ZPLs are seen in this region much less frequently. Spectral jumps of ZPLs were observed. The dynamics as well as the spectral range covered by such jumps differ for C708 and C715/C719. This heterogeneity is likely caused by differences in the close environment of the chromophores. A tentative assignment of C708 and C715/C719 to Chla dimers and a Chla trimer is discussed, which is based on the remarkable structural differences in the environment of the most probable candidates for the red-most fluorescence.

[NiFe] hydrogenases contain a highly conserved histidine residue close to the [NiFe] active site which is altered by a glutamine residue in the H(2)-sensing [NiFe] hydrogenases. In this study, we exchanged the respective glutamine residue of the H(2) sensor (RH) of Ralstonia eutropha, Q67 of the RH large subunit HoxC, by histidine, asparagine and glutamate. The replacement by histidine and asparagine resulted in slightly unstable RH proteins which were hardly affected in their regulatory and enzymatic properties. The exchange to glutamate led to a completely unstable RH protein. The purified wild-type RH and the mutant protein with the Gln/His exchange were analysed by continuous-wave and pulsed electron paramagnetic resonance (EPR) techniques. We observed a coupling of a nitrogen nucleus with the [NiFe] active site for the mutant protein which was absent in the spectrum of the wild-type RH. A combination of theoretical calculations with the experimental data provided an explanation for the observed coupling. It is shown that the coupling is due to the formation of a weak hydrogen bond between the protonated N(epsilon) nucleus of the histidine with the sulfur of a conserved cysteine residue which coordinates the metal atoms of the [NiFe] active site as a bridging ligand. The effect of this hydrogen bond on the local structure of the [NiFe] active site is discussed.

In this study we examined the energy transfer dynamics of a FRET coupled pair of chromophores at the single molecule level embedded in a tunable sub-wavelength Fabry-Pérot resonator with two silver mirrors and separations in the λ/2 region. By varying the spectral mode density in the resonator via the mirror separation we altered the radiative relaxation properties of the single chromophores and thus the FRET efficiency. We were able to achieve wavelength dependent enhancement factors of up to three for the spontaneous emission rate of the chromophores while the quenching due to the metal surfaces was nearly constant. We could show by confocal spectroscopy, time correlated single photon counting and time domain rate equation modeling that the FRET rate constant is not altered by our resonator.