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

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 ν_CO band at 1890 cm^–1 and ν_CN 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 ν_CO band and 2047 and 2061 cm^–1 ν_CN 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 ν_CO and ν_CN 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 Δ<i>H</i> (6.4 ± 0.8 kJ mol^–1) and Δ<i>S</i> (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