Hydroxy-bridged resting states of a [NiFe]-hydrogenase unraveled by cryogenic vibrational spectroscopy and DFT computations

Abstract

Active site vibrations of a [NiFe]-hydrogenase catalytic subunit are selectively probed by IR and NRV spectroscopy in two NiIIFeII and NiIIIFeII resting states, contributing in combination with DFT modeling to rationalized structural candidates.

Exploring Structure and Function of Redox Intermediates in [NiFe]‐Hydrogenases by an Advanced Experimental Approach for Solvated, Lyophilized and Crystallized Metalloenzymes

“…In order to specifically probe the metal‐bound hydride of the Ni a ‐C state, lyophilized Re RH samples were treated with both H 2 and D 2 (Figure 4 B ). The resulting experimental spectra were compared to simulated data obtained by DFT calculations (Figure 4 B ), based on an Re RH active‐site homology model[ 42 , 43 ] (Figures S5 and S6; for details, see the DFT Methods section of the Supporting Information). Analysis of the Ni a ‐C spectrum obtained after incubation of Re RH with H 2 (Figure 4 B , top blue trace) revealed two intense bands at 554 and 598 cm −1 and a weaker one at 574 cm −1 .…”

Optimization of Culture Conditions for Oxygen-Tolerant Regulatory [NiFe]-Hydrogenase Production from Ralstonia eutropha H16 in Escherichia coli

“…Infrared (IR) spectroscopy was used to probe the ν(CO) (1870-2020/cm) and ν(CN) (2030-2150/cm) stretching modes of the CO and CN − ligands of the [NiFe] catalytic center, which are observed in a spectral region devoid of protein contribution. Homologously produced HoxC exhibits two CO bands at 1941/cm and 1952/cm and broad CN − signals at 2066/cm and 2084/cm, which are related to the two Ni r -S I/II resting states of the catalytic center (Figure 7A) [14,18]. The IR spectrum of native RH stop , on the other hand, exhibits a dominant CO band at 1943/cm and two CN − absorptions at 2070/cm and 2081/cm, which have been previously assigned to the catalytic Ni a -S intermediate (Figure 7B) [34].…”

“…In addition to the RH heterodimer, consisting of the HoxB and HoxC subunits, the H 2 -sensing two-component regulatory system includes the histidine kinase HoxJ and the response regulator HoxA [11] (Figure 1). Considering the high complexity of the RH system depicted in Figure 1, many in vitro studies, e.g., EPR [12], IR [13][14][15], Mössbauer [16], resonance Raman (RR) [17], and nuclear resonance vibrational (NRVS) [18] spectroscopy, have been carried out on a truncated version of the protein (RH stop , Figure 1 right side), which allows for the isolation of the single HoxBC heterodimer [19,20]. Nevertheless, relatively low protein yields of 0.01-0.1 mg of purified enzyme per L of culture have been reported for both homologous (R. eutropha) [13,19,20] and heterologous (E. coli) [21] RH stop production.…”

Ein neuer Aufbau zur Untersuchung der Struktur und Funktion von solvatisierten, lyophilisierten und kristallinen Metalloenzymen – veranschaulicht anhand von [NiFe]‐Hydrogenasen

“…Die hierfür durchgeführten Rechnungen basieren auf einem Homologiemodell des aktiven Zentrums der ReRH (Abbildungen S5 und S6;siehe DFT-Methodenteil der Supporting Information fürD etails). [42,43] Das Ni a -C-Spektrum der H 2 -reduzierten ReRH (blau in Abbildung 4B,o ben) zeigt zwei intensive Banden bei 554 und 598 cm À1 und eine weniger intensive bei 574 cm À1 .Z usätzliche Signale sind bei 446, 470, 500 und 508 cm À1 erkennbar.S chwingungsspektroskopische Signale von [NiFe]-Hydrogenasen in diesem Frequenzbereich wur-den bisher Normalmoden zugeordnet, die grçßtenteils Fe-CO-und Fe-CN-Biege-und Streckschwingungen enthalten. [19][20][21][25][26][27][28] Insgesamt ist die Übereinstimmung zwischen den experimentellen und berechneten Spektren gut (Abbildung S7).…”

“…Die zugehörigen experimentellen Spektren wurden mit den entsprechenden DFT‐Daten verglichen (Abbildung 4 B). Die hierfür durchgeführten Rechnungen basieren auf einem Homologiemodell des aktiven Zentrums der Re RH (Abbildungen S5 und S6; siehe DFT‐Methodenteil der Supporting Information für Details) [42, 43] . Das Ni a ‐C‐Spektrum der H 2 ‐reduzierten Re RH (blau in Abbildung 4 B, oben) zeigt zwei intensive Banden bei 554 und 598 cm −1 und eine weniger intensive bei 574 cm −1 .…”