Article pubs.acs.org/JPCB
FT-IR Characterization of the Light-Induced Ni-L2 and Ni-L3 States of [NiFe] Hydrogenase from Desulfovibrio vulgaris Miyazaki F Hulin Tai,†,‡ Koji Nishikawa,§ Seiya Inoue,§ Yoshiki Higuchi,‡,§ and Shun Hirota*,†,‡ †
Graduate School of Materials Science, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma-shi, Nara 630-0192, Japan ‡ CREST, Japan Science and Technology Agency, Gobancho, Chiyoda-ku, Tokyo 102-0076, Japan § Graduate School of Life Science, University of Hyogo, 3-2-1 Koto kamigori-cho, Ako-gun, Hyogo 678-1297, Japan S Supporting Information *
ABSTRACT: Different light-induced Ni-L states of [NiFe] hydrogenase from its Ni-C state have previously been observed by EPR spectroscopy. Herein, we succeeded in detecting simultaneously two Ni-L states of [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F by FT-IR spectroscopy. A new light-induced νCO band at 1890 cm−1 and νCN bands at 2034 and 2047 cm−1 were detected in the FT-IR spectra of the H2-activated enzyme under N2 atmosphere at basic conditions, in addition to the 1910 cm−1 νCO band and 2047 and 2061 cm−1 νCN bands of the Ni-L2 state. The new bands were attributed to the Ni-L3 state by comparison of the FT-IR and EPR spectra. The νCO and νCN frequencies of the Ni-L3 state are the lowest frequencies observed among the corresponding frequencies of standard-type [NiFe] hydrogenases in various redox states. These results indicate that a residue, presumably Ni-coordinating Cys546, is protonated and deprotonated in the Ni-L2 and Ni-L3 states, respectively. Relatively small ΔH (6.4 ± 0.8 kJ mol−1) and ΔS (25.5 ± 10.3 J mol−1 K−1) values were obtained for the conversion from the Ni-L2 to Ni-L3 state, which was in agreement with the previous proposals that deprotonation of Cys546 is important for the catalytic reaction of the enzyme. wing to its reversible H2 oxidation reaction, H2 ⇌ 2H+ + 2e−, hydrogenase is one of the most highly studied enzymes, with promising applications in future energy technologies.1−6 Knowledge of the reaction mechanism of hydrogenase will provide notable information for utilizing hydrogen bacteria as well as developing artificial compounds. In nature, there are three types of hydrogenases: [NiFe], [FeFe], and [Fe] hydrogenases.7−9 The most well-studied hydrogenase is the standard-type [NiFe] hydrogenase. [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F (DvMF) consists of two subunits: large and small.10,11 The catalytic center of [NiFe] hydrogenase from DvMF is located in its large subunit and contains two metal ions, Ni and Fe, bridged with two cysteinyl thiolates (Cys84, Cys549) (Figure 1A). Another two cysteine residues (Cys81, Cys546) are terminally bound to the Ni ion, whereas three diatomic ligands (one CO and two CN−) are coordinated to the Fe ion.10−13 Three Fe−S clusters are located in the small subunit of [NiFe] hydrogenase. The electrons obtained by the heterolytic cleavage of H2 at the catalytic Ni− Fe center are transferred through these Fe−S clusters to the physiological redox partner, cytochrome c3.14,15 The “as-isolated” oxidized state of [NiFe] hydrogenase is a mixture of two paramagnetic Ni-A (unready) and Ni-B (ready) states (Ni3+), where an oxygenic ligand is bridged between the Ni and Fe ions in both states.16−19 An EPR-silent state (Ni-SIa,
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© 2015 American Chemical Society
Figure 1. (A) Active site structure of H2-activated [NiFe] hydrogenase from DvMF (PDB: 1H2R) and (B) its proposed catalytic cycle.
Ni2+) is produced through the EPR-silent Ni-SU and Ni-SIr states (Ni2+) by activation (reduction) of the Ni-A and Ni-B states, respectively, and a paramagnetic state (Ni-C, Ni3+) and fully reduced EPR-silent state (Ni-R, Ni2+) are generated by further reduction.2−6 The Ni-SIa, Ni-C, and Ni-R states are reported to be involved in the catalytic cycle of [NiFe] Special Issue: Wolfgang Lubitz Festschrift Received: March 31, 2015 Revised: April 20, 2015 Published: April 21, 2015 13668
DOI: 10.1021/acs.jpcb.5b03075 J. Phys. Chem. B 2015, 119, 13668−13674
Article
The Journal of Physical Chemistry B hydrogenase.20,21 These states convert among each other by addition or release of protons, electrons, and H2 (Figure 1B).2−6,20,21 A bridging hydride (H−) between the Ni and Fe ions has been suggested for the Ni-C state by Lubitz and others, using electron nuclear double resonance (ENDOR) spectroscopy22 and theoretical calculations.23−25 Reduction of the Ni-C state lead to formation of the Ni-R state, indicating the presence of the hydride also in the Ni-R state.26−28 Recently, Ogata et al. have reported a ultrahigh resolution X-ray structure of the Ni-R state of anaerobically isolated [NiFe] hydrogenase from DvMF at 0.89 Å resolution.29 The hydride at the Ni−Fe bridge and a proton bound to the sulfur atom of Nicoordinating Cys546 were observed in the X-ray structure, indicating Cys546 as an initial proton acceptor for the heterolytic cleavage of H2. The Ni-C state is converted to another paramagnetic Ni-L state (Ni+) by light irradiation at low temperatures.30−36 Absorption bands were observed at ∼590 and ∼700 nm for the Ni-C state, and the photoconversion of the Ni-C state to the Ni-L state was observed throughout the visible region with local maxima at ∼590 and ∼700 nm.30 Conversion of the Ni-C state to the Ni-L state is associated with a loss of the hydride between the Ni and Fe ions and transfer of the produced proton from the Ni−Fe site to Cys546, according to ENDOR and hyperfine sublevel correlation measurements together with theoretical studies.22,23,37 However, two or three Ni-L states (Ni-L1, Ni-L2, and Ni-L3) have been observed in the EPR spectra of various standard-type [NiFe] hydrogenases by light irradiation of the Ni-C state under anaerobic conditions at
