80 Chemical Science Hydrogenases (H 2 ases) are enzymes that catalyze both the production and consumption of molecular hydrogen [1,2]. They play an important role in biology, allowing organisms to efficiently use protons as electron acceptors and evolving H 2 , and also allowing capture of H 2 in their role as ‘uptake H 2 ases’ [3]. They are also of interest for potential applications in a hydrogen economy, either used directly or serving as inspirational targets for biomimetic inorganic catalysts [4]. A detailed knowledge of the catalytic mechanisms of H 2 ases is thus an important goal for biology, chemistry, and clean energy technology. In the [NiFe] family of H 2 ases, catalysis takes place at a Ni-Fe dimetallic center (Fig. 1). In this unique active site, Fe is linked to Ni by a pair of cysteine thiolate ligands. Depending on the redox poise or degree of aerobic deactivation, the Fe and Ni centers may also be bridged by hydride, oxo/hydroxo, and possibly hydroperoxo ligands. The Fe site also carries one CO and a pair of CN – ligands. [NiFe] H 2 ases also possess additional Fe-S clusters that relay electrons to and from the active site. Although crystallography has revealed many essential features about the NiFe active site, there are additional details that are more easily addressed by spectroscopy. For example, infrared spectroscopic studies have revealed more than ten different chemical species, using the CO and CN – stretching bands in the 1800 – 2100 cm –1 region. Only a few forms of the enzyme can be crystallized as homogeneous chemical species, and even then, it is hard to see all of the details by crystallography with atomic resolution. The alternate species often differ by the bridge between Ni and Fe, which is often hard to see clearly in the crystallographic data. In the oxidized, inactive forms Ni-A and Ni-B, it is proposed that Ni and Fe are bridged by O 2– , OH – or even OOH – species, while the reduced and active forms ‘Ni-C’ and ‘Ni-R’ are both thought to possess a hydride (H – ) bridge. However, the critical hydride bridge is not visible in the crystal structures. In the current study [5], we employed a relatively new technique called Nuclear Resonance Vibrational Spectroscopy (NRVS), which is only sensitive to motion of 57 Fe, to identify H 2 ase Fe- CN and Fe-CO modes in the 400 – 600 cm –1 region. Such studies, combined with measurement of model compounds and Density Functional Theory (DFT) calculations will help characterize the geometry of the bridges to iron and the nature of hydrogen activation. NRVS has evolved into a powerful technique for probing the dynamics of Fe in metalloproteins. The measurement employs scanning a highly monochromatic (meV) X-ray beam through a nuclear resonance. Unlike Mössbauer spectroscopy, which detects ‘recoil-free’ nuclear transitions, NRVS detects the inelastic events that involve creation or destruction of phonons. The resulting spectrum is similar to a conventional IR or Raman spectrum, but with different selection rules. The NRVS intensity for a given normal mode is related to the amount of motion of the resonant nucleus (in this case 57 Fe) in that mode. One can therefore observe only the 57 Fe-based vibrations for a protein sample involving thousands of other atoms. The measurements were conducted at beamline BL09XU . As seen in Fig. 2, the NRVS of H 2 ase is dominated by contributions from the Fe-S clusters in the electron transport chain. The structure around 150 cm –1 derives from cluster breathing and bending modes, while the features between 200 and 400 cm –1 are mostly Fe-S stretching modes. Fortunately, the Fe-S cluster NRVS dies out rapidly above 400 cm –1 , and at A dynamic view of [NiFe] hydrogenase by means of nuclear resonance vibrational spectroscopy (c) (e) Large subunit Ni-Fe Proximal [4Fe4S] Medial [3Fe4S] Distal [4Fe4S] Small subunit Cys216 Cys222 Cys191 Cys150 Cys114 Cys20 Cys17 His188 His88 CN – CN – Ni Fe CO Ser502 Arg479 (a) (b) (d) Cys249 Cys231 Cys252 Fig. 1. The structures of the Fe sites in H 2 ase. (a) overall view of the electron transport chain. (b-e) detailed views of individual clusters, including: (b) ‘distal’ [4Fe-4S] cluster, showing Cys 3 His ligation, (c) ‘medial’ [3Fe-4S] cluster, (d) ‘proximal’ [4Fe-4S], and (e) Ni-Fe active site. The atoms are Fe (brown), Ni (green), S (yellow), C (gray), N (blue) and O (red). 81 still higher frequencies it is still possible to distinguish Fe-CN and Fe-CO stretching and bending modes from the active site Fe. Upon reduction of the protein, we were able to see modest upshifts in the Fe-CO stretch that correlated with shifts seen in the mid-IR CO stretching bands. Going forward, a truly exciting prospect of this work is the potential for observation of Fe hydride/ deuteride stretching and bending modes. In recent work on a Ni-H/D-Fe model compound (Fig. 3 ), we found that weak Fe-H stretches could be seen past 1500 cm –1 , while much stronger Fe-H-Ni bending modes were visible near 800 cm –1 . Finding the position of these bands in H 2 ase will provide valuable information about the nature of hydrogen activation at the active site. The continuing improvement in the emittance of the SPring-8 storage ring, the brightness available from improved undulators, and the stability of high-resolution X-ray monochromators have all contributed to make this work possible. 0 200 300 100 400 500 600 57 Fe PVDOS (cm) Energy (cm –1 ) Energy (cm –1 ) 400 500 600 (a) (b) (c) 542 547 563 605 621 562/566 [NiFe] center of Hydrogenase [Fe(bdt)(CN) 2 CO ] 2– S S S S S S X C C N N N N Ni C C C C O O Fe Fe Fig. 2. NRVS-derived PVDOS for Fe sites in H 2 ase. Left: overall spectra for oxidized ( –– ) vs reduced ( –– ) H 2 ase. Right: close-ups for Fe-CO/CN region. Top to bottom: (a) (NEt 4 ) 2 [ 57 Fe(bdt)(CN) 2 (CO)] ( –– ) and (NEt 4 ) 2 [ 57 Fe(tdt) (CN) 2 (CO)] ( –– ), (b) oxidized H 2 ase, and (c) reduced H 2 ase. 800 1000 1200 1400 1600 Energy (cm –1 ) 'Ni- stretch' × 3 Ni-H-Fe bend Fe-D stretch Fe-H stretch × 15 × 5 S P H Ni CO Fe 'N i-Dstretch' × 3 Ni-H-Fe bend F Fe-D D stretch Fe-H stretch × 15 × 5 S P P H H Ni O CO F Fe Fig. 3. Recent NRVS data on [(CO) 3 57 Fe(pdt)H/DNi(dppe)]BF 4 (blue for H and red for D) - a Ni-Fe model for NiFe H 2 ase. The model structure is as shown in the insert. H. Wang a,c , H. Ogata b , W. Lubitz b, * and S. P. Cramer a,c, * a Department of Chemistry, University of California, USA b Max Planck Institute for Chemical Energy Conversion, Germany c Physical Biosciences Division, Lawrence Berkeley National Laboratory, USA *Email: wolfgang.lubitz@cec.mpg.de, spjcramer@ucdavis.edu References [1] P.M. Vignais et al. : FEMS Microbiol. Rev. 25 (2001) 455. [2] F.A. Armstrong: Curr. Opin. Chem. Biol. 8 (2004) 133. [3] I. Valdez-Vazquez, H.M. Poggi-Varaldo: Renew. Sust. Energy Rev. 13 (2009) 1000. [4] C. Tard, C.J. Pickett: Chem. Rev. 109 (2009) 2245. [5] S. Kamali, H. Wang, D. Mitra, H. Ogata, W. Lubitz, B.C. Manor, T.B. Rauchfuss, D. Byrne, V. Bonnefoy, F.E. Jenney Jr., M.W.W. Adams, Y. Yoda, E. Alp, J. Zhao and S.P. Cramer: Angew. Chem. Int. Edit. 52 (2013) 469.
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