Photoactivation of the Ni-SIr state to the Ni-SIa state in [NiFe] hydrogenase: FT-IR study on the light reactivity of the ready Ni-SIr state and as-isolated enzyme revisite

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(1)Please do not adjust margins. Journal Name COMMUNICATION. Received 00th January 20xx, Accepted 00th January 20xx. Photoactivation of the Ni-SIr state to Ni-SIa state in [NiFe] hydrogenase: FT-IR study on the light reactivity of the ready Ni-SIr state and as-isolated enzyme revisited†. DOI: 10.1039/x0xx00000x. Hulin Tai,a,b Liyang Xu,a Seiya Inoue,c Koji Nishikawa,c Yoshiki Higuchi,b,c and Shun Hirotaa,b,*. www.rsc.org/. The Ni-SIr state of [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F was photoactivated to its Ni-SIa state by Ar+ laser irradiation at 514.5 nm, whereas the Ni-SL state was light induced from a newly identified state, which was less active than any other identified state and existed in the “as-isolated” enzyme.. Introduction Hydrogenase is a metalloenzyme which catalyzes the reversible H2 oxidation reaction, H2 ⇌ 2H+ + 2e−.1-4 According to the active site metal composition, hydrogenases are classified into three types: [NiFe], [FeFe], and [Fe].4 [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F (DvMF) is a membrane-attached enzyme comprising two subunits, one large and one small.5-7 The large subunit contains the Ni-Fe active site, where the Ni and Fe ions are bridged with two cysteinyl thiolates (Fig. 1). Another two cysteine residues are terminally bound to the Ni ion, whereas one CO and two CN− ligands are coordinated to the Fe ion.7-10 The small subunit contains three Fe-S clusters which mediate the electron transfer between the Ni−Fe active site and cytochrome c3.11 Aerobically isolated [NiFe] hydrogenase, herein referred to as “asisolated”, is a mixture of mainly two paramagnetic Ni-A (Ni3+) and Ni-B (Ni3+) states with some other EPR-silent states.10,12,13 The NiB state is readily activated in the presence of H2 or under electrochemically reducing conditions, while the Ni-A state requires longer time for activation.14,15 A bridging hydroxidoo (OH−) ligand between the Ni and Fe ions has been identified for the Ni-B state (Fig.1).6,16 For the Ni-A state, the nature of an oxygenic bridging ligand remains contentious,6,16-21 however, bridging OH− and cysteine-sulfenate ligands between the Ni and Fe ions have been indicated recently.22 One electron reduction of the Ni-A and Ni-B states produces EPR-silent unready Ni-SU and ready Ni-SIr states (Ni2+), respectively.10,16 The Ni-SIr state is activated into another EPR-silent Ni-SIa state (Ni2+) by protonation at the Ni−Fe active site. through an acid−base equilibrium, where the Ni-SIr and Ni-SIa states represent the deprotonated and protonated states, respectively.10,12,23 Several mechanisms have been proposed to explain the acid−base equilibrium. In one of them, the bridging OH− ligand is present in the Ni-SIr state, and a proton is transferred to the OH− ligand, which then leaves the active site as a H2O molecule.2,12,24,25 In the other proposals, a bridging OH− ligand may be present, absent, or replaced by a hydride (H−) or a H2O molecule in the Ni-SIr state, and the proton is transferred to one of the terminal Ni-coordinating cysteinethiolate26,27 or cysteine-sulfenate28 ligand that acts as a proton accepting base in the Ni-SIr state. The acid−base equilibrium between the Ni-SIr and Ni-SIa states is a common feature among [NiFe] hyrogenases, and thus the Ni-SIr state has been identified as a key intermediate for the enzyme activation.4 Further reduction of the Ni-SIa state produces a paramagnetic state (Ni-C, Ni3+) and a fully reduced EPR-silent state (Ni-R, Ni2+), where the Ni-SIa, Ni-C, and Ni-R states form a catalytic cycle.3,4,10 Light sensitivity of [NiFe] hydrogenase has been reported for various states and utilized to elucidate its catalytic reaction.29-36 For example, we have reported photo-conversion of the Ni-C state to the Ni-L and Ni-SIa states for DvMF [NiFe] hydrogenase, and proposed the Ni-L state as an intermediate between the transition of the Ni-C and Ni-SIa states.31 The Ni-L state has also been shown to be a catalytic intermediate for [NiFe] hydrogenases from Pyrococcus furiosus and Escherichia coli by chemical potential jump kinetic and direct electrochemical studies.37-40 We have also simultaneously detected two Ni-L states (Ni-L2 and Ni-L3) by FT-IR, and proposed that Ni-coordinating Cys546 is deprotonated during the conversion from the Ni-L2 to Ni-L3 state.32 Furthermore, it has been proposed that the Ni-SIr state is light sensitive, reversibly forming an EPRCys84. Cys81. Ni. CN− O. Fe CO. a. Graduate School of Materials Science, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma-shi, Nara 630-0192, Japan E-mail: hirota@ms.naist.jp; FAX: +81-743-72-6119; Tel: +81-743-72-6110 b CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan c Graduate School of Life Science, University of Hyogo, 3-2-1 Koto kamigori-cho, Ako-gun, Hyogo 678-1297, Japan †Electronic Supplementary Information (ESI) available. See DOI: 10.1039/x0xx00000x. Cys546. CN− Cys549. Fig. 1 Active site structure of DvMF [NiFe] hydrogenase in the Ni-B state (PDB: 1WUJ). One CO and two CN− ligands are assigned as Fe ligands.7,16,23 Carbon, nitrogen, oxygen, sulphur, nickel, and iron atoms are shown in grey, blue, red, yellow, green, and pink spheres, respectively.. J. Name., 2016, 00, 1-3 | 1. This journal is © The Royal Society of Chemistry 20xx. Please do not adjust margins.

(2) Please do not adjust margins COMMUNICATION. Journal Name. silent Ni-SL state (Ni2+) at 90−110 K.36 However, in this work, we found that the Ni-SL state is not light induced from the Ni-SIr state, but rather the Ni-SIr state is photo-induced to the Ni-SIa state.. Experimental. Ni-SIr 1924. 0.005. a. 2071 2056. b. Preparation of [NiFe] hydrogenase [NiFe] hydrogenase was isolated from sulfate reducing bacterium DvMF, and purified as described previously.5 The concentration of [NiFe] hydrogenase was adjusted with its absorption at 400 nm using its absorption coefficient (ε = 47 mM-1cm-1).11 FT-IR measurements [NiFe] hydrogenase (concentration 1.0–2.0 mM) in 25 mM Tris-HCl buffer (pH 7.4 at 298 K) was degassed with a vacuum line, purged with 1 bar of H2, and incubated at 310 K for 5.5 h (if not mentioned) to obtain the H2-activated sample. The sample solution was further degassed with the vacuum line and purged with 1 bar of N2. The NiSIr state was obtained by partial oxidation of the H2-activated enzyme with an anaerobic addition of 5 equivalents of phenosafranin (Sigma-Aldrich) using a glove box (YSD-800L, UNICO, Tsukuba). The sample solution was transferred anaerobically into an infrared cell with CaF2 windows in the glove box. FT-IR spectra were measured before, during, and after light irradiation at 103–238 K with a FT-IR spectrometer (FT-IR 6100V, JASCO, Tokyo) equipped with an MCT detector. A cryostat system (CoolSpeK IR USP203IR-A, Unisoku, Hirakata) was used to control the temperature of the cell. The light irradiation spectra were measured 5–22 min after light-irradiation was started. Light irradiation of the sample was performed at 514.5 nm with an Ar+ laser (Model 2017, SpectraPhysics, Santa Clara). The laser power was adjusted to 0.5–3.3 W/cm2 at the sample point. The corresponding buffer spectrum was collected as a reference spectrum and subtracted from the sample spectra. Spectral data were collected at 2-cm-1 resolution and averaged from 1024 scans.. Results and discussion Observation of the light-induced states at low temperatures It has been reported that the midpoint potential (Em) for the redox transition between the Ni-B and Ni-SI (Ni-SIr and Ni-SIa) states of DvMF [NiFe] hydrogenase is −151 mV at pH 7.4, whereas between the Ni-SI and Ni-C states it is −375 mV.23 Under N2 atmosphere, the H2-activated enzyme contained the Ni-C and Ni-R states for ~70% and ~30%, respectively (See S1, ESI†), with ~90 % of the proximal Fe-S cluster reduced.29 The Ni-SIr state was obtained by partial oxidation of the H2-activated enzyme with an anaerobic addition of 5 equivalents of phenosafranin under N2 atmosphere, since phenosafranin exhibits its redox potential at Em = −252 mV between −375 and −151 mV. The CO stretching (νCO) and CN− stretching (νCN) frequencies of the Fe site are reliable sensors for the changes in the electron density of the Fe ion in [NiFe] hydrogenase.41 Negative IR bands at 1924, 2056, and 2071 cm-1 and positive bands at 1943, 2077, and 2089 cm1 were observed in the difference (light-minus-before) FT-IR spectra between the spectra during and before light irradiation by Ar+ laser (514.5 nm) for phenosafranin-oxidized [NiFe] hydrogenase at 178– 238 K under N2 atmosphere at pH 8.0 (Fig. 2A). The negative and. A. 238 K. ×3. Ni-SIa 1943. 238 K. a. 198 K Ni-AL 1972. 2076 2090. B Ni-SX 1922. 2070 2061. b. 2077 2089. Ni-A and Ni-B 1957. 0.005. Ni-SL Ni-A 1968 1957. 198 K 158 K. ×3. 218 K. c. d. ×3. 198 K. d. 123 K. e. ×3. 178 K. e. 103 K. c. 2100. 2000. 1900. 2100. 2000. 1900. Fig. 2 FT-IR spectra of (A) phenosafranin-oxidized and (B) as-isolated DvMF [NiFe] hydrogenase at 178–238 and 103–198 K, respectively, under N2 atmosphere at pH 8.0. (a) FT-IR spectra before light irradiation and (b–e) light-minus-before difference spectra between the spectra during and before light irradiation are shown. Phenosafranin-oxidized enzyme was obtained by partial oxidation of the H2-activated enzyme with an anaerobic addition of 5 equivalents of phenosafranin. The difference spectra of phenosafranin-oxidized enzyme are expanded by three. The laser power was adjusted to 2.5 W/cm2 at the sample point. The pH value was measured at 274 K.. positive bands were related to the light-sensitive reactant and lightinduced product, respectively. The frequency of the negative band at 1924 cm-1 corresponded to that of the νCO band of the Ni-SIr state of DvMF [NiFe] hydrogenase, whereas 2056 and 2071 cm-1 corresponded well to the frequencies of its conjugated νCN bands.23 The positive frequencies at 1943, 2077, and 2089 cm-1 corresponded well to those of the νCO and two conjugated νCN bands of the Ni-SIa state of the H2-activated enzyme.23,31,32 These results reveal that the Ni-SIr state sonnverts to the Ni-SIa state by the light irradiation (Fig. 3). Ciaccafava et al. have reported that electrochemical activation of an O2-tolerant [NiFe] hydrogenase from Aquifex aeolicus is promoted by UV-vis light irradiation, but the detailed activation mechanism was unspecified.30 Although the Ni-SIr state has not been observed by electrochemical FT-IR measurements for O2-tolerant [NiFe] hydrogenases, the Ni-SIr state may be highly reactive leading to the fast transition of the Ni-B state to Ni-SIa state by the light irradiation.42 Judging from the intensities of the νCO bands of the NiSIr state in the light-minus-before difference FT-IR spectra, approximately 3% of the Ni-SIr state was converted to the Ni-SIa state by the light irradiation at 238 K. The intensities of the νCO bands of the Ni-SIr and Ni-SIa states increased in the light-minusbefore difference spectra with a decrease in the temperature, and approximately 34% of the Ni-SIr state was converted to the Ni-SIa state at 178 K. Notably, the light-induced conversion of the Ni-SIr state decreased significantly at pH 9.6 (See S2, ESI†), indicating that protonation occurred in the photoactivation process. The reported photochemical reactions in various [NiFe] hydrogenases are usually associated with dissociation of non-protein ligands bound to the metal ions at the Ni−Fe active site.13,29-36,43 Stronger laser power was required for photoactivation of the Ni-SIr state to the Ni-SIa state compared to photoactivation of the Ni-C state to the NI-L state associated with dissociation of the bridging H−.31 Considering these results, we propose that the protonation of the Ni-SIr state is related to dissociation of the putative bridging OH− ligand as a H2O molecule by the light irradiation, although the possibility of the Ni-. 2 | J. Name., 2016, 00, 1-3. This journal is © The Royal Society of Chemistry 20xx. Please do not adjust margins.

(3) Please do not adjust margins Journal Name. COMMUNICATION. Ni3+,. Fe2+. 1956, 2085, 2094. Ni-SL Ni2+, Fe2+. (1968, 2076, 2090). hν. e−, H+. Ni-SX Ni-SU Ni2+, Fe2+. Ni2+, Fe2+. (1922, 2061, 2070) 1958, 2089, 2100. Ni3+,. Fe2+. 1955, 2081, 2090. e−. Ni-SIr. H+,. hν. Ni2+, Fe2+. Ni+, Fe2+ n.d.. −H2O. e−, H+. (1924, 2056, 2071). Ni-CO. Ni-SIa 1943. A. Ni-B. CO. Active CO. Ni-SIa Ni2+, Fe2+. (1943, 2077, 2089). Ni-L. H2. Ni-SCO Ni2+, Fe2+. 1941, 2056, 2071, 2084. Ni-R. Ni+, Fe2+. (1911, 2048, 2062). Ni2+, Fe2+. 1948, 2061, 2074. Ni-C. Ni3+, Fe2+. 2074 2071. 2087. a. 2057. Ni-C 1961. Ni-SIr 1923. Ni-SCO 1941. 2084. 2056 (νCO) b. b. Fig. 3 Reaction states of [NiFe] hydrogenase. The νCO and νCN frequencies for each state of DvMF [NiFe] hydrogenase are depicted.13 The frequencies of the Ni-CO state has not been identified for DvMF [NiFe] hydrogenase. The frequencies at 198 and 298 K are shown with and without parentheses, respectively.. coordinating cysteine-thiolate or cysteine-sulfenate ligand being protonated after the light irradiation cannot be excluded.26-28 The light-induced FT-IR spectrum converted back immediately to the initial spectrum when the light irradiation was stopped at 218 and 238 K, and no νCO or νCN band was observed in the difference (afterminus-before) FT-IR spectra between the spectra after and before light irradiation (See S3, ESI†). However, the νCO and νCN bands of the Ni-SIa state were observed in the after-minus-before difference spectrum at 198 K and the intensities in the difference spectrum increased at 178 K, indicating that the light-induced Ni-SIa state was trapped at low temperatures. Interestingly, the relative intensities of the νCO bands of the Ni-SIr and Ni-SIa states in the light-minusbefore difference spectra at 103 K depended on the irradiation time and light intensity (See S4, ESI†). The Ni-SIr state photo-converted to the Ni-SIa state for only ~50 % after ~40 min of light irradiation with the laser power of 3.3 W/cm2 at 103 K and pH 8.0, indicating that the photo-conversion process of the Ni-SIr state to the Ni-SIa state was relatively slow. However, the photo-conversion rate of the Ni-SIr state did not change significantly at pH 7.0 compared to that at pH 8.0, although the relative intensity of the Ni-SIr νCO band before light irradiation decreased. A negative IR band at 1957 cm-1 and a positive band at 1972 cm-1 were observed in the light-minus-before difference FT-IR spectrum of as-isolated DvMF [NiFe] hydrogenase under N2 atmosphere at pH 8.0 and 198 K (Fig. 2B). The negative and positive bands corresponded well to the νCO bands of the Ni-A and light-induced Ni-AL states, respectively.29,36 Additionally, negative IR bands at 1922, 2061, and 2070 cm-1 and positive bands at 1968, 2076, and 2090 cm-1 were observed in the light-minus-before difference spectra at 103–158 K. The frequencies of the positive bands were similar to the νCO and νCN frequencies of the light-induced Ni-SL state obtained from as-isolated DvMF [NiFe] hydrogenase,36 indicating formation of the Ni-SL state. However, the frequencies of the negative bands (νCO: 1922 cm-1, νCN: 2061 and 2070 cm-1) were 1–5 cm-1 shifted from those of the νCO and two conjugated νCN bands of the Ni-SIr state (νCO: 1924 cm-1, νCN: 2056 and 2071 cm-1) (Fig. 2).23 These frequency differences indicate that the light-reacted state other than the Ni-SIr state converted to the Ni-SL state. We term this newly identified light-reacted state, Ni-SX (Fig. 3). Previously, spectroscopic studies on the as-isolated DvMF [NiFe] hydrogenase have determined a third bridging ligand between the Ni and Fe ions in addition to two cysteinyl thiolates has been suggested by. Ni-SX 1922. a. 2071. e−, H+. 1961, 2074, 2087. Ni-A and Ni-B 1956. B. Normalized absorbance. Ni-A. Normalized absorbance. Inactive. 2100. 2000 1900 Wa en mber (cm 1). 2100. 2000 1900 Wa en mber (cm 1). spectroscopic studies for a photoactive state of as-isolated DvMF [NiFe] hydrogenase, but the state was misassigned to the Ni-SIr state.36 The Ni-SX state corresponds to this previously studied state. The intensities of the νCO and νCN bands of the Ni-SX and Ni-SL states in the difference Fig. 4 FT-IR spectra of (A) phenosafranin-oxidized and (B) as-isolated DvMF [NiFe] hydrogenase under (a) N2 and (b) CO atmospheres at pH 7.4 and 298 K.. (light-minus-after) spectra between the spectra during and after light irradiation increased with decrease in the temperature from 158 K to 103 K, indicating reversibility for the formation of the Ni-SL state by the light irradiation (See S5, ESI†). Differences between the Ni-SIr and Ni-SX states in pH sensitivity and carbon monoxide reactivity The acid−base equilibrium between the Ni-SIr and Ni-SIa states is important for the activation of [NiFe] hydorogenase (Fig. 3).4 In the FT-IR spectrum of phenosafranin-oxidized [NiFe] hydrogenase under N2 atmosphere at pH 7.4 and 298 K, the νCO bands of the NiSIr (1923 cm-1; corresponding to the 1924-cm-1 band at 178–238 K in Fig. 2A) and Ni-SIa (1943 cm-1) states were the major νCO bands, whereas a weak νCO band (1961 cm-1) corresponding to the Ni-C state was also detectable (See S6A, ESI†). The pH-dependence of the ratio between the Ni-SIa and Ni-SIr νCO band intensities revealed that the Ni-SIa and Ni-SIr states form an acid−base equilibrium.10,12,23 In the spectrum of as-isolated [NiFe] hydrogenase, the νCO bands corresponding to the Ni-A (1956 cm-1), Ni-B (1955 cm-1), and Ni-SX (1922 cm-1) states were observed,13,23,36 but the intensities of these bands did not change with a change in pH (See S6B, ESI†), showing that no acid−base equilibrium existed for the Ni-SX state. These results support the hypothesis that the Ni-SX state is different from the Ni-SIr state and indicate that the Ni-SX state is not a ready state. Carbon monoxide (CO) is known as a reversible inhibitor for [NiFe] hydrogenases from earlier enzymatic studies.44 X-ray crystallographic experiments have demonstrated that exogenous CO coordinates to the active site Ni ion of DvMF [NiFe] hydrogenase.43 For the FT-IR spectrum of phenosafranin-oxidized [NiFe] hydrogenase in CO-saturated buffer, IR bands were observed mainly at 1941, 2056, 2071, and 2084 cm-1 (Fig. 4A). The bands at 1941, 2071 and 2084 cm-1 correspond well to the νCO and two conjugated νCN bands of the Ni-SCO state (exogenous CO-bound state), whereas the band at 2056 cm-1 corresponds well to that of the exogenous CO bound to the Ni ion, revealing that most of the enzyme molecules were in the Ni-SCO state.13 Previous spectroscopic studies have showed that CO reacts selectively with the Ni-SIa and Ni-L. J. Name., 2016, 00, 1-3 | 3. This journal is © The Royal Society of Chemistry 20xx. Please do not adjust margins.

(4) Please do not adjust margins COMMUNICATION. Journal Name. states.13,36,43-45 These results indicate that the Ni-SIr state was converted into the Ni-SCO state under CO atmosphere apparently through the acid−base equilibrium (Fig. 3). On the other hand, no change was observed in the FT-IR spectrum of as-isolated [NiFe] hydrogenase by introduction of CO, indicating that the Ni-SX state Ni-A and Ni-B 1957 Ni-SX 1922. Ni-SL 1968. a b. Ni-C 1963. Ni-R 1951. c d. e f. site.22 The required reduction of the persulfide bond may explain the observed slow activation of the Ni-SX state. As-isolated DvMF [NiFe] hydrogenase contained the Ni-SX state exhibiting a νCO band at 1922 cm-1 and two νCN bands at 2061 and 2070 cm-1.13,36 The intensities of these bands decreased exponentially with a time constant of ~50 min by incubation under H2 atmosphere at 310 K (See Figs. S8 and S9, ESI†), revealing that the Ni-SX state was activated very slowly under H2 atmosphere. However, the Ni-SX state was not observed for the air-oxidized enzyme, where the H2-activated enzyme was exposed to air (Fig. 5, curves e and f). Additionally, the Ni-SX state was not observed after further dithionite reduction of the air-oxidized enzyme (Fig. 5, curves g and h). These results reveal that although the as-isolated enzyme contained the Ni-SX state, the Ni-SX state was not formed during the generation or activation of the Ni-A and Ni-B states in vitro.. g. Conclusions. h 2100. 2000. 1900. Fig. 5 FT-IR spectra of DvMF [NiFe] hydrogenase under N2 atmosphere at pH 8.0 and 103 K: (a,c,e,g) Spectra before light irradiation and (b,d,f,h) lightminus-after difference spectra. The spectra of the as-isolated enzyme in the (a,b) absence and (c,d) presence of 10 equivalents of dithionite and the spectra of the air-oxidized enzyme (obtained by exposure of the H2activated enzyme to air) in the (e,f) absence and (g,h) presence of 10 equivalents of dithionite are shown. The “after” spectra were measured 30– 47 min after light irradiation was stopped. The laser power was adjusted to 2.5 W/cm2 at the sample point. The pH value was measured at 274 K.. was unreactive toward CO (Fig. 4B), in agreement with the results that an acid−base equilibrium did not exist for the Ni-SX state (See S6B, ESI†). Reaction of the Ni-SX state with dithionite To gain information on the activation of the Ni-SX state, lightminus-after difference FT-IR spectra were measured for as-isolated and dithionite-reduced DvMF [NiFe] hydrogenase at 103 K (Fig. 5). The photo-conversion of the Ni-SX state to the Ni-SL state was successfully observed in the light-minus-after difference spectra, because the Ni-SL state converted back to the Ni-SX state (See S5, ESI†) but the light-induced Ni-SIa and Ni-L states were trapped at 103 K when the light irradiation was stopped (See S7, ESI†).31 By assuming equal absorption coefficients for the νCO bands, approximately 16% was in the Ni-SX state for the as-isolated enzyme (Fig. 5, curve a), and almost (~95%) all the enzymes in the Ni-SX state converted to the Ni-SL state by the light irradiation at 103K (Fig. 5, curve b). However, approximately 10% of the enzyme was still in the Ni-SX state after reduction with dithionite (Fig. 5, curves c and d), indicating that the Ni-SX state was very inactive. For [NiFe] hydrogenases from the sulphur-metabolizing bacterium Allochromatium vinosum and Desulfovibrio fructosorans (Df), an inactive state (Ni-‘Sox’) similar to the Ni-SX state has been reported.12,22 The νCO and νCN frequencies of the Ni-‘Sox’ state for Df [NiFe] hydrogenase (νCO: 1911, νCN: 2059 and 2068 cm-1) were 1–5 cm-1 shifted from those of its Ni-SIr state (νCO: 1913 cm-1, νCN: 2054 and 2069 cm-1),22,46 which was very similar to those of the Ni-SX state detected in the present study (Fig. 2B). The Ni-‘Sox’ state has been proposed by X-ray crystallographic analysis to possess a cysteine-persulfide terminal Ni-coordinating ligand at the active. We have shown for the first time that the ready Ni-SIr state of DvMF [NiFe] hydrogenase is converted to the active Ni-SIa state by laser light irradiation at 514.5 nm (Fig. 3). From the pH-dependent lightreactivity of the Ni-SIr state, we propose that the bridging OH− ligand dissociates as a H2O molecule from the Ni−Fe active site by light irradiation at low pH. We have identified a light-sensitive NiSX state (νCO, 1922 cm-1; νCN, 2061 and 2070 cm-1), which was photo-converted to the Ni-SL state. A certain amount of the enzyme was still in the Ni-SX state after treatment of the as-isolated enzyme with dithionite, although the enzyme was activated slowly by H2, revealing that the Ni-SX state was highly inactive. These findings provide new insights into the activation mechanism of [NiFe] hydrogenase.. Acknowledgment We thank Prof. Takashi Matsuo, Nara Institute of Science and Technology, for the use of the glove box system and helpful discussions. We also acknowledge Mr. Leigh McDowell for his advice during manuscript preparation. 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(6) Supplementary Information. Photoactivation of the Ni-SIr state to Ni-SIa state in [NiFe] hydrogenase: FT-IR study on the light reactivity of the ready Ni-SIr state and as-isolated enzyme revisited Hulin Tai,a,b Liyang Xu,a Seiya Inoue,c Koji Nishikawa,c Yoshiki Higuchi,b,c and Shun Hirotaa,b,* a. Graduate School of Materials Science, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma-shi, Nara 630-0192, Japan, bCREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan, and cGraduate School of Life Science, University of Hyogo, 3-2-1 Koto kamigori-cho, Ako-gun, Hyogo 678-1297, Japan *E-mail: hirota@ms.naist.jp; Fax: +81-743-72-6119; Phone: +81-743-72-6110 Contents Fig. S1. FT-IR spectra of H2-activated and phenosafranin-oxidized [NiFe] hydrogenase.. p. S2. Fig. S2. FT-IR spectra of phenosafranin-oxidized [NiFe] hydrogenase before light irradiation and their light-minus-before difference spectra at pH 9.6.. p. S3. Fig. S3. FT-IR spectra of phenosafranin-oxidized [NiFe] hydrogenase before light irradiation and their light-minus-before and after-minus-before difference spectra at different temperatures (178−238 K).. p. S4. Fig. S4. FT-IR spectra of phenosafranin-oxidized [NiFe] hydrogenase before light irradiation and their light-minus-before difference spectra with different irradiation time.. p. S5. Fig. S5. FT-IR spectra of as-isolated [NiFe] hydrogenase before light irradiation and their light-minus-after difference spectra at different temperatures (103−198 K).. p. S6. Fig. S6. FT-IR spectra of phenosafranin-oxidized and as-isolated [NiFe] hydrogenase at different pH values.. p. S7. Fig. S7. FT-IR spectra of H2-activated [NiFe] hydrogenase before and after weak light irradiation and difference spectrum between the spectra during strong light irradiation and after weak light irradiation.. p. S8. Fig. S8. FT-IR spectra of as-isolated and H2-treated [NiFe] hydrogenase before light irradiation and their light-minus-after difference spectra.. p. S9. Fig. S9. H2 activation kinetics of the Ni-SX state.. p. S10. S1.

(7) Normalized absorbance. Ni-C 1962 Ni-R 1948 a Ni-SIa 1943 Ni-SIr 1923 b 2100. 2000 1900 -1 Wavenumber (cm ). Fig. S1 FT-IR spectra of (a) H2-activated and (b) phenosafranin-oxidized DvMF [NiFe] hydrogenase under N2 atmosphere at pH 7.4 and 298 K. Phenosafranin-oxidized [NiFe] hydrogenase was obtained by partial oxidation of the H2-activated enzyme with an anaerobic addition of 5 equivalents of phenosafranin under N2 atmosphere. The pH value was measured at 298 K. The νCO frequency of the Ni-SIr state at 298 K was shifted to a lower frequency for about 1 cm-1 compared to that obtained at 178–238 K, although the frequency shift was smaller than the resolution (2 cm-1).. S2.

(8) Ni-SIr 1924 a. 2071 2056. b. 218 K. 2077. c. 218 K. ×3. 2089. ×3. Ni-SIa 1943. 198 K. d. ×3. 178 K. e. ×3. 158 K. 2100. 2000 1900 -1 Wavenumber (cm ). Fig. S2 FT-IR spectra of phenosafranin-oxidized DvMF [NiFe] hydrogenase (a) before light irradiation at 218 K and (b–e) light-minus-before difference spectra between the spectra during and before light irradiation at 158–218 K under N2 atmosphere at pH 9.6. Phenosafranin-oxidized [NiFe] hydrogenase was obtained by partial oxidation of the H2-activated enzyme with an anaerobic addition of 5 equivalents of phenosafranin under N2 atmosphere. The laser power was adjusted to 2.5 W/cm2 at the sample point. The pH value was measured at 274 K.. S3.

(9) A. 0.005. Ni-SIr 1924. 2071 2056. B. 0.002. Ni-SIa 1943. 238 K. 2089. 238 K. 218 K. 2077. 198 K. 218 K. 178 K 198 K. 2056 2071. Ni-SIr 1924. 178 K. 2100. 2000 1900 Wavenumber (cm-1). 2100. 2000 1900 Wavenumber (cm-1). Fig. S3 FT-IR spectra of phenosafranin-oxidized DvMF [NiFe] hydrogenase before light irradiation and their light-minus-before and after-minus-before difference spectra at different temperatures (178, 198, 218, and 238 K) under N2 atmosphere at pH 8.0. (A) FT-IR spectra before light irradiation and (B) light-minus-before difference spectra between the spectra during and before light irradiation (black), and after-minus-before difference spectra between after and before light irradiation (magenta and cyan) are shown. The “after” spectra were measured 5–22 min (magenta) and 30–47 min (cyan) after light irradiation was stopped. Phenosafranin-oxidized [NiFe] hydrogenase was obtained by partial oxidation of the H2-activated enzyme with an anaerobic addition of 5 equivalents of phenosafranin under N2 atmosphere. The laser power was adjusted to 2.5 W/cm2 at the sample point. The pH value was measured at 274 K.. S4.

(10) A. Ni-SIr 1924. a b. Ni-SIr 1924. B a. Ni-SIa 1943. C a. Ni-SIa 1943. b. b. c. c. c. d. d. d. e. e. e. f. f. f. g. g. g. h. h. h. 2100 2000 1900 Wavenumber (cm -1). Ni-SIr 1924. 2100 2000 1900 Wavenumber (cm -1). Ni-SIa 1943. 2100 2000 1900 Wavenumber (cm -1). Fig. S4 FT-IR spectra of phenosafranin-oxidized DvMF [NiFe] hydrogenase before light irradiation and their light-minus-before difference spectra under N2 atmosphere at 103 K with different irradiation time and light intensity at (A) pH 7.0 and (B,C) pH 8.0. (a) FT-IR spectra before light irradiation and (b-h) light-minus-before difference spectra between the spectra during and before light irradiation are shown. The laser power was adjusted to (A,C) 3.3 and (B) 1.3 W/cm2 at the sample point. The light-irradiation spectra were measured (b) 3–20, (c) 30–47, (d) 60– 57, (e) 90–107, (f) 120–137, (g) 150–167, and (h) 180–197 min after light irradiation was started. Phenosafranin-oxidized [NiFe] hydrogenase was obtained by partial oxidation of the H2-activated enzyme with an anaerobic addition of 5 equivalents of phenosafranin under N2 atmosphere. The pH value was measured at 274 K.. S5.

(11) A. 0.005. Ni-A and Ni-B 1957. B. 0.005. 2087 2092. 2082. 2097. 2070 2061. 198 K. Ni-SX 1922 198 K. 2090 2076. Ni-SL 1968. 158 K. Ni-AL 1972. 158 K. 123 K. 123 K. 103 K 2061 2070. Ni-SX 1922. 103 K. 2100. 2000 1900 -1 Wavenumber (cm ). 2100. 2000 1900 -1 Wavenumber (cm ). Fig. S5 FT-IR spectra of as-isolated DvMF [NiFe] hydrogenase before light irradiation and their light-minus-after difference spectra at different temperatures (103, 123, 158, and 198 K) under N2 atmosphere at pH 8.0. (A) FT-IR spectra before light irradiation and (B) light-minus-after difference spectra between the spectra during and after light irradiation. The “after” spectra were measured 5–22 min after light irradiation was stopped. The laser power was adjusted to 2.5 W/cm2 at the sample point. The pH value was measured at 274 K.. S6.

(12) A. pH9.0. Ni-SIa 1943. Ni-SIr 1923 Normalized absorbance. 2071 2075. Normalized absorbance. Ni-A and Ni-B 1956. B. 2087 2056. pH8.0. Ni-C 1962. pH7.4 2100. pH9.0. Ni-SX 1922. pH7.4. pH6.0 2000 1900 Wavenumber (cm-1). 2100. 2000 1900 Wavenumber (cm-1). Fig. S6 FT-IR spectra of DvMF [NiFe] hydrogenase under N2 atmosphere at different pH values (pH 6.0, 7.4, 8.0, and 9.0) at 298 K: (A) Phenosafranin-oxidized and (B) as-isolated [NiFe] hydrogenase. Phenosafranin-oxidized [NiFe] hydrogenase was obtained by partial oxidation of the H2-activated enzyme with an anaerobic addition of 5 equivalents of phenosafranin under N2 atmosphere. The pH value was measured at 298 K.. S7.

(13) Ni-C. 0.005. 1963. Ni-R Ni-SIa. 2077, 2077 2071. a. 1951. 1943. Ni-SIr. 1923 Ni-L2 1910. 2063 2089 2056 2062 2048. b. c. 2100. 2000 1900 -1 Wavenumber (cm ). Fig. S7 FT-IR spectra of H2-activated DvMF [NiFe] hydrogenase before and after weak light irradiation and difference spectrum between the spectra during strong light irradiation and after week light irradiation under N2 atmosphere at pH 8.0 and 103 K: (a) Before light irradiation, (b) after weak light irradiation, and (c) difference spectra between the spectra during strong light irradiation and after weak light irradiation. The weak and strong laser powers were adjusted to 0.5 and 2.5 W/cm2, respectively, at the sample point. The “after” spectrum with weak light irradiation was measured 5–22 min after light irradiation was stopped. The pH value was measured at 274 K. No light-induced conversion of the Ni-SX state to Ni-SL state was observed in the difference spectrum.. S8.

(14) A. B. Ni-A and Ni-B 1957. Ni-C 1963. Ni-SX. Ni-SL 1968. 1922. a Ni-R 1950. Nomalized Δabsorbance. Nomalized absorbance. a Ni-SIr and Ni-SX 1923. b. c. b. c Ni-SX 1922 d. d 2100. 2000 1900 -1 Wavenumber (cm ). 2100. 2000 1900 -1 Wavenumber (cm ). Fig. S8 FT-IR spectra of DvMF [NiFe] hydrogenase (A) before light irradiation and (B) their lightminus-after difference spectra between the spectra during and after light irradiation under N2 atmosphere at pH 8.0 and 103 K: (a) as-isolated and (b) 30 min, (c) 60 min, and (d) 120 min H2treated [NiFe] hydrogenase. To obtain the 30 min, 60 min, and 120 min H2-treated [NiFe] hydrogenase, the as-isolated enzyme solution was degassed with a vacuum line, purged with 1 bar of H2, and incubated at 310 K for (b) 30, (c) 60, and (d) 120 min, respectively. The “after” spectra were measured 30–47 min after light irradiation was stopped. The laser power was adjusted to 2.5 W/cm2 at the sample point. The pH value was measured at 274 K.. S9.

(15) Normalized intensity of Ni-SX νCO band. 1. 0 0. 300 100 200 H2 treated time (min). Fig. S9 H2 activation kinetics of the Ni-SX state at 310 K. Intensities of the Ni-SX νCO band were calculated from Figures S7 and S8. The intensity of the νCO band decreased exponentially with a time constant of ~50 min.. S10.

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