Relativistic DFT Calculations of the Paramagnetic Intermediates of [NiFe] Hydrogenase. Implications for the Enzymatic Mechanism Matthias Stein, Erik van Lenthe, † Evert Jan Baerends, † and Wolfgang Lubitz* Max-Volmer-Institut fu ¨ r Biophysikalische Chemie und Biochemie, Technische UniVersita ¨ t Berlin Strasse des 17. Juni 135, 10623 Berlin, Germany ReceiVed NoVember 20, 2000 ReVised Manuscript ReceiVed February 26, 2001 Hydrogenases are enzymes that catalyze the reversible hetero- lytic dissociation of molecular hydrogen H 2 a H + + H - . The largest class of hydrogenases contains a NiFe center that is believed to be the catalytic site for hydrogen activation. 1 Recent insight into the structure of the active site has come from X-ray structure analyses of single crystals of the [NiFe] hydrogenases from DesulfoVibrio (D.) gigas 2 and D. Vulgaris Miyazaki F. 3 The active site (Figure 1) comprises a heterobimetallic cluster of Ni and Fe atoms. The bridging ligand X was proposed to be an oxygen or sulfur species in the oxidized states of D. gigas and D. Vulgaris, respectively; X was found to be absent in the crystal structure of the reduced state of two enzymes. 4 Three nonprotein diatomics (2 CN and 1 CO) ligate the Fe atom. 5 The “as-isolated” oxidized state of the [NiFe] hydrogenase is a mixture of two paramagnetic forms (Ni-A and Ni-B) with slightly different g-values. 1 Ni-B (or “ready”) is reduced within minutes under an H 2 atmosphere while Ni-A (or “unready”) requires incubation for several hours. An EPR-silent state (Ni- Si) is passed before a third paramagnetic state (Ni-C) and the fully reduced state (Ni-R) is obtained. Ni-C is believed to be an intermediate in the catalytic cycle. Upon illumination, the Ni-C state is converted into a fourth paramagnetic state (Ni- L). Carbon monoxide is an inhibitor of the enzyme yielding a paramagnetic CO-bound state (Ni-CO). All paramagnetic states are S ) 1 / 2 . Previous quantum mechanical studies have addressed the question of H 2 activation by [NiFe] hydrogenases 6 and were mainly used to calculate IR transitions. 7 Here, we present the first relativistic 8 description and calculation of magnetic resonance parameters (g-tensors) of a transition metal containing enzyme 9 and show that these values can be correlated with structural parameters. This approach allows us to propose a reaction mechanism for the [NiFe] hydrogenases. Ni-B/Ni-A: The g-tensor magnitudes and orientations of the oxidized states were determined from EPR investigations of single crystals. 10 From the similarities of the g-values of Ni-A (2.32, 2.24, 2.01) and Ni-B (2.33, 2.16, 2.01), a drastic change in the electronic structure of the active site in the Ni-A state compared to Ni-B is unlikely. The g-tensor orientation was found to be very similar for Ni-B and Ni-A. 10 In the calculations, first the possibility of a sulfur species 3 (i.e. S 2- , SH - , or H 2 S) as bridging ligand X (Figure 1) was considered but did not lead to satisfying results. 11 Next, an oxygenic species was considered as X. 12 Such a species was postulated to occupy the position of the bridging ligand in D. gigas 2b and A. Vinosum. 13 The g-tensor orientation and the principal values of Ni-B were confirmed by our calculations when a OH - ligand occupies the position of the bridging ligand (Table 1). 14 The calculated Ni-Fe distance is 3.00 Å, in reasonable agreement with the value obtained from the X-ray analysis of D. gigas (2.9 Å). ZORA calculations with a depro- tonated bridging ligand, i.e., a O 2- bridge, gave g-values of 2.36, 1.95, 1.84. It was investigated whether the unrealistic values below g e result from the spin-restricted nature of the wave function. 15,16 The consideration of spin-polarization drastically improved the description. Thus a µ-oxo bridge was assigned to Ni-A. This is supported by the absence of D 2 O exchangeable protons in the active center of the Ni-A state 17 and the required prolonged exposure to H 2 to be activated compared to Ni-B (see below). Protonation of a terminal cysteine as proposed in ref 7b did not yield satisfying g-values. 18 The postulated protonation of the O 2- bridge in the Ni-B form would not be detectable in the X-ray structure due to the limited resolution of 2.5 Å. 2b † Theoretical Chemistry, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands. (1) Albracht, S. P. J. Biochim. Biophys. Acta 1994, 1188, 167-204. (2) (a) Volbeda, A.; Charon, M.-H.; Piras, C.; Hatchikian, E. C.; Frey, M.; Fontecilla-Camps, J.-C. Nature 1995, 373, 580-587. (b) Volbeda, A.; Garcin, E.; Piras, C.; de Lacey, A. L.; Fernandez, V. M.; Hatchikian, E. C.; Frey, M.; Fontecilla-Camps, J. C. J. Am. Chem. Soc. 1996, 118, 12989-12996. (3) Higuchi, Y.; Yagi, T.; Yasuoka, N. Structure 1997, 5, 1671-1680. (4) (a) Higuchi, Y.; Ogata, H.; Miki, K.; Yasuoka, N.; Yagi, T. Structure 1999, 7, 549-556. (b) Garcin, E.; Vernede, X.; Hatchikian, E. C.; Volbeda, A.; Frey, M.; Fontecilla-Camps, J.-C. Structure 1999, 7, 557-566. (5) (a) Happe, R. P.; Roseboom, W.; Pierik, A. J.; Albracht, S. P. J. Nature 1997, 385, 126. (b) De Lacey, A. L.; Hatchikian, E. C.; Volbeda, A.; Frey, M.; Fontecilla-Camps, J.-C.; Fernandez, V. M. J. Am. Chem. Soc. 1997, 119, 7181-7189. (6) (a) Pavlov, M.; Siegbahn, P. E. M.; Blomberg, M. R. A.; Crabtree, R. H. J. Am. Chem. Soc. 1998, 120, 548-555. (b) DeGioia, L.; Fantucci, P.; Guigliarelli, B.; Bertrand, P. Inorg. Chem. 1999, 38, 2658-2662. (c) Pavlov, M.; Blomberg, M. R. A.; Siegbahn, P. E. M. Int. J. Quantum Chem. 1999, 73, 197-207. (d) DeGioia, L.; Fantucci, P.; Guigliarelli, B.; Bertrand, P. Int. J. Quantum Chem. 1999, 73, 187-195. (7) (a) Niu, S.; Thomson, L. M.; Hall, M. B. J. Am. Chem. Soc. 1999, 121, 4000-4007. (b) Amara, P.; Volbeda, A.; Fontecilla-Camps, J.-C.; Field, M. J. J. Am. Chem. Soc. 1999, 121, 4468-4477. (8) (a) van Lenthe, E.; Wormer, P. E. S.; van der Avoird, A. J. Chem. Phys. 1997, 107, 2488-2498. (b) van Lenthe, E.; van der Avoird, A.; Wormer, P. E. S. J. Chem. Phys. 1998, 108, 4783-4796. (9) Computational details: The ZORA formalism as implemented in the Amsterdam Density Functional (ADF) package was used. The calculations are single-point calculations at nonrelativistic geometry-optimized structures using the BP86 exchange-correlation functional. Cysteines were modeled as CH3-CH2-S - groups. No constraints were imposed on the structures. A double- Slater-type basis set with polarization functions (basis II in ADF nomenclature) was used. A triple- basis set is used for the 3d shells of the first transition metals. The following orbitals were frozen during geometry optimizations: C 1s, N 1s, O 1s, S up to 2p, Ni up to 2p, Fe up to 2p. The calculations of magnetic resonance parameters were performed in an all- electron basis. (10) (a) Gessner, C.; Trofanchuk, O.; Kawagoe, K.; Higuchi, Y.; Yasuoka, Y.; Lubitz, W. Chem. Phys. Lett. 1996, 256, 518-524. (b) Trofanchuk, O.; Stein, M.; Gessner, C.; Lendzian, F.; Higuchi, Y.; Lubitz, W. J. Biol. Inorg. Chem. 2000, 5, 36-44. (11) After complete geometry optimizations the Ni-Fe distances are 3.15 Å for H2S, 3.11 Å for SH - , and 3.19 Å for S 2- , whereas the X-ray structures yield 2.55 Å for D. Vulgaris Miyazaki F and 2.90 Å for D. gigas. The calculated g-values (gx, gy, gz) are 2.19, 2.06, 2.01 for a H2S, 2.19, 2.15, 1.99 for a SH - , and 2.31, 2.07, 1.91 for an S 2- ligand. These are not in good agreement with experimental values. (12) Comparison of structural data from X-ray analysis and geometry optimizations are available as Supporting Information. (13) Van der Zwaan, J. W.; Coremans, J. M. C. C.; Bouwens, E. C. M.; Albracht, S. P. J. Biochim. Biophys. Acta 1990, 1041, 101-110. (14) The deviation of the calculated gx-value from the experimental value is not unusual for the ZORA approach (Stein, M.; van Lenthe, E.; Baerends, E. J.; Lubitz, W. J. Phys. Chem. A 2001, 105, 416-425). Figure 1. Active site of [NiFe] hydrogenase from D. gigas (predomi- nantely in the Ni-A oxidation state) 2 , Cys ) cysteine. 5839 J. Am. Chem. Soc. 2001, 123, 5839-5840 10.1021/ja005808y CCC: $20.00 © 2001 American Chemical Society Published on Web 05/26/2001