A strenuous experimental journey searching for spectroscopic evidence of a bridging nickel–iron–hydride in [NiFe] hydrogenase

Abstract

Direct spectroscopic evidence for a hydride bridge in the Ni-R form of [NiFe] hydrogenase has been obtained using iron-specific nuclear resonance vibrational spectroscopy (NRVS). The Ni-H-Fe wag mode at 675 cm À1 is the first spectroscopic evidence for a bridging hydride in Ni-R as well as the first ironhydride-related NRVS feature observed for a biological system. Although density function theory (DFT) calculation assisted the determination of the Ni-R structure, it did not predict the Ni-H-Fe wag mode at $ 675 cm À1 before NRVS. Instead, the observed Ni-H-Fe mode provided a critical reference for the DFT calculations. While the overall science about Ni-R is presented and discussed elsewhere, this article focuses on the long and strenuous experimental journey to search for and experimentally identify the Ni-H-Fe wag mode in a Ni-R sample. As a methodology, the results presented here will go beyond Ni-R and hydrogenase research and will also be of interest to other scientists who use synchrotron radiation for measuring dilute samples or weak spectroscopic features.

Proton Transfer in the Catalytic Cycle of [NiFe] Hydrogenases: Insight from Vibrational Spectroscopy

“…Experimental evidence for this representation of Ni a -R and the presence of a bridging hydride ligand was first provided by a 0.89 Å resolution crystal structure of the [NiFe] hydrogenase from D. vulgaris MF (electron density map shown in Figure 4 B), 11 where a shortened Ni–H distance relative to Fe–H showed tighter binding of the hydride to Ni at the active site. The presence of a bridging hydride was confirmed by nuclear resonance vibrational spectroscopy (NRVS) 94 , 95 where a vibration at 675 cm –1 was shown to be sensitive to H/D isotope exchange and assigned to a Ni–H–Fe wag vibrational mode by comparison with DFT calculations and spectra of model compounds ( Figure 4 C). Protonation of a terminal thiolate is supported by earlier DFT calculations on the EPR silent states of the active site; thiolate protonation was required to model the Ni a -SI state, and both thiolate protonation and a bridging hydride ligand were necessary to accurately reproduce the IR peak positions of the Ni a -R I state of D. vulgaris MF [NiFe] hydrogenase.…”

Hydrogenase Enzymes and Their Synthetic Models: The Role of Metal Hydrides

“…89 The hydrido binds Ni (1.58 Å) somewhat tighter than Fe (1.78 Å), and its presence has since been verified by 57 Fe NRVS, with the observation of a characteristic δ NiHFe bending vibration at 675 cm −1 . 103,312 …”

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

“…Die NRVS‐Daten in Abbildung 4 zeigen den für die Fe‐CO/CN‐Moden (400–650 cm −1 ) und die Ni‐H‐Fe‐Wippschwingungen (650–800 cm −1 ) charakteristischen Spektralbereich [25–28] . Die vom Detektor aufgezeichnete Zählrate am elastischen Peak erhöhte sich für die lyophilisierte Probe um den Faktor vier im Vergleich zur Lösungsprobe (rechts oben in Abbildung 4 A), was zu einer deutlichen Verbesserung des Signal‐Rausch‐Verhältnisses führte.…”

“…Dabei werden die fürE isen spezifischen Normalmoden selektiv detektiert. [25][26][27][28][29][30][31][32] Im Rahmen dieser Arbeit haben wir einen experimentellen Aufbau zur spektroskopischen Analyse von gasumwandelnden Metalloenzymen in verschiedenen Probenformen entwickelt. Durch die Einstellung der Gaszusammensetzung und Te mperatur kçnnen spezifisch Redoxzustände präpariert und anschließend durch komplementäre spektroskopische Methoden untersucht werden.…”