Matthias Stein

Single crystal EPR studies of the reduced active site of [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F.

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.

A single-crystal ENDOR and density functional theory study of the oxidized states of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F.

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 β-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.

Quantum chemical calculations of [NiFe] hydrogenase.

During the past five years, the metalloenzyme [NiFe] hydrogenase has increasingly attracted the interest of quantum chemists. In particular, different approaches have been applied to investigate the mechanism of the heterolytic splitting of molecular hydrogen. These theoretical studies have stimulated many new questions rather than given final answers. It has recently become possible to directly calculate experimental observables from first principles. Here, it is demonstrated how vibrational frequencies, g-values and hyperfine coupling constants (for Fourier-transformed infrared spectroscopy and electron paramagnetic resonance spectroscopy) can be obtained, which allow a direct comparison with experimental data.

DFT calculations of the electronic structure of the paramagnetic states Ni-A, Ni-B and Ni-C of [NiFe] hydrogenase

Structural n parameters and atomic spin densities of the active centre of [NiFe] hydrogenases in the paramagnetic n states Ni-A, Ni-B and Ni-C are reported. The DFT (BLYP/DZVP) calculated spin density distribution is n found to be in good agreement with experimental data when the bridging ligand is OH− in the Ni-B and O2− n in the n Ni-A states of the enzyme. For the reduced enzyme (Ni-C) it is postulated that a hydride ligand bridges the n Ni and Fe atoms. The atomic spin densities for the proposed paramagnetic states are in good agreement n with experimental electron magnetic resonance data. Based on the deduced composition of n the states Ni-A, n Ni-B and Ni-C a model for n the reaction mechanism is proposed.

The electronic structure of the catalytic intermediate Ni-C in [NiFe] and [NiFeSe] hydrogenases

The catalytic intermediate Ni-C in the heterolytic cleavage of molecular hydrogen is studied using relativistic DFT calculations. The difference between the [NiFe] and [NiFeSe] hydrogenases is investigated and it is found that structural modifications lead to changes in the electronic structure, manifested by g-values and hyperfine interactions. The sulfur to selenium substitution leads to small changes in the structural parameters and the spin density distribution. The g-tensor principal values are changed but the g-tensor orientation is retained in both enzymes. The hyperfine parameters show small but systematic changes. Participation of the terminal cysteine Cys530 in the reaction mechanism is discussed.

A theoretical study of spin states in Ni-S4 complexes and models of the [NiFe] hydrogenase active site

We have applied density functional theory, using both pure (BP86) and hybrid (B3LYP and B3LYP*) functionals, to investigate structural parameters and reaction energies for nickel(II)-sulfur coordination compounds, as well as for small cluster models of the Ni-SI and Ni-R redox state of [NiFe] hydrogenases. Results obtained investigating experimentally well-characterized complexes show that BP86 is well suited to describe the structural features of this class of compounds. However, the singlet–triplet energy splitting and even the computed ground state are strongly dependent on the applied functional. Results for the cluster models of [NiFe] hydrogenases lead to the conclusion that in the reduced protein structures characterized by X-ray diffraction a hydride bridges the two metal centres. The energy splitting of the singlet and triplet states in Ni-R and Ni-SI models is calculated to be very small and may be overcome at room temperature to allow a spin crossover. Moreover, the relative stability of the Ni-SI and Ni-R structures adopted in the present investigation is fully compatible with their involvement in the reversible heterolytic cleavage of H2.

High-frequency EPR studies on cofactor radicals in photosystem I

Electron paramagnetic resonance (EPR) spectroscopy at W-band (94 GHz) is used to resolve theg-tensors of the radical ions of the primary chlorophyll donor P700+⋅ and the quinone acceptor A1−⋅ in photosystem I. The obtainedg-tensor principal values are compared with those of the isolated pigment radicals in organic solvents and the shifts are related to an impact of the protein environment. P700+⋅ has been investigated in photosystem I single crystals at 94 GHz. W-band EPR applied to the photoinduced radical pair P700+⋅A1−⋅ is used to correctly assign theg-tensor axes of P700+⋅ to the molecular structure of the primary donor. Density functional theory calculations on a model of A1−⋅ in its binding pocket derived from the recent crystal structure of photosystem I were utilized to correlate experimental magnetic resonance parameters with structural elements of the protein.

Orientation-selected ENDOR of the active center in Chromatium vinosum [NiFe] hydrogenase in the oxidized "ready" state.

Abstractu2002Electron nuclear double resonance (ENDOR) was applied to study the active site of the oxidized ready state, Nir, in the [NiFe] hydrogenase of Chromatium vinosum. The magnetic field dependence of the EPR was used to select specific subsets of molecules contributing to the ENDOR response by stepping through the EPR envelope. Three hyperfine couplings could be clearly followed over the complete field range. Two protons, H1 and H2, display a very similar large isotropic coupling of 12.5 and 12.6u2009MHz, respectively. Their dipolar coupling is small (2.1 and 1.4u2009MHz, respectively). A third proton, H3, exhibits a small isotropic coupling of 0.5u2009MHz and a larger anisotropic contribution of 3.5u2009MHz. Based on a comparison with structural data obtained from X-ray crystallography of single crystals of hydrogenases from Desulfovibrio gigas and D. vulgaris and the known g-tensor orientation of Nir, an assignment of the 1H hyperfine couplings could be achieved. H1 and H2 were assigned to the β-CH2 protons of the bridging cysteine Cys533 and H3 could belong to a β-CH2 proton of Cys68 or to a protonated cysteine (-SH) of Cys68 or Cys530.