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 v(CO) band at 1890 cm(-1) and v(CN) bands at 2034 and 2047 cm(-1) were detected in the FT-IR spectra of the H-2-activated enzyme under N-2 atmosphere at basic conditions, in addition to the 1910 cm-1 v(CO) band and 2047 and 2061 cm-1 v(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 v(CO) and v(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 Delta H (6.4 +/- 0.8 kJ mol(-1)) and Delta S (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.