The large subunit of the regulatory [NiFe]-hydrogenase fromRalstonia eutropha– a minimal hydrogenase?

Abstract

Spectroscopic investigation of an isolated [NiFe]-hydrogenase large subunit enables a unique view of the NiFe(CO)(CN)2 cofactor.

Hydroxy-bridged Active Site States of [NiFe]-Hydrogenase Unraveled by Cryogenic Vibrational Spectroscopy and DFT Computations

“…1,[24][25][26] The IR spectrum of HoxCox, recorded at room temperature, displayed bands assigned to Nir-B-like species termed as Nir-B´ and Nir-B´´, respectively. 19 Low-temperature IR measurements performed on HoxCox revealed the Nir-B´´ state, characterized by a CO absorption at 1979 cm -1 , as the predominant species at 85 K (Fig. 2b).…”

“…12,14,18 Recently, we isolated the catalytic subunit HoxC from the O2-tolerant regulatory [NiFe]hydrogenase (RH) of Ralstonia eutropha. 19 HoxC hosts exclusively the [NiFe] active site, and it is therefore an ideal target for a detailed and exclusive spectroscopic investigation of the catalytic center without interference from the auxiliary FeS clusters. In the present study, we applied low-temperature IR and NRVS techniques together with density functional theory (DFT) calculations to gain structural information of so-far barely defined hydroxy-bridged active site states of [NiFe]-hydrogenase.…”

“…1 and 2a). 1,19 A previous study showed that these states can be partially interconverted by shifting the pH of the buffer. 19 This has been interpreted with the presence of a protonatable bridging ligand at the active site, attributed to a hydroxy (OH -) group (Fig.…”

“…In our previous work, we showed that HoxCai protein can be oxidized with ferricyanide, revealing a species denoted as HoxCox. 19 According to X-ray, EPR and DFT studies, standard…”

“…S3). 19 The fact that almost pure Nir-SI and Nir-B´´ states were observed in HoxCai and HoxCox, respectively, facilitated the establishment of DFT models, which were subsequently used to interpret the low-temperature IR and NRV spectra. (Fig.…”