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J Phys Chem B. 2015 Oct 29;119(43):13668-74. doi: 10.1021/acs.jpcb.5b03075. Epub 2015 Apr 30.

FT-IR Characterization of the Light-Induced Ni-L2 and Ni-L3 States of [NiFe] Hydrogenase from Desulfovibrio vulgaris Miyazaki F.

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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.


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.

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