Caught in the H_inact: Crystal Structure and Spectroscopy Reveal a Sulfur Bound to the Active Site of an O₂‐stable State of [FeFe] Hydrogenase

Abstract

[FeFe] hydrogenases are the most active H2 converting catalysts in nature, but their extreme oxygen sensitivity limits their use in technological applications. The [FeFe] hydrogenases from sulfate reducing bacteria can be purified in an O2‐stable state called Hinact. To date, the structure and mechanism of formation of Hinact remain unknown. Our 1.65 Å crystal structure of this state reveals a sulfur ligand bound to the open coordination site. Furthermore, in‐depth spectroscopic characterization by X‐ray absorption spectroscopy (XAS), nuclear resonance vibrational spectroscopy (NRVS), resonance Raman (RR) spectroscopy and infrared (IR) spectroscopy, together with hybrid quantum mechanical and molecular mechanical (QM/MM) calculations, provide detailed chemical insight into the Hinact state and its mechanism of formation. This may facilitate the design of O2‐stable hydrogenases and molecular catalysts.

A Beginner’s Guide to Thermodynamic Modelling of [FeFe] Hydrogenase

“…Using this simple model, we can plot the dependence of each of the species Ox, Red, OxH, and RedH as a function of either the applied potential at a given pH ( Figure 4A) or as a function of the pH at a given applied potential ( Figure 4B). 7, (11), (12), and (15), with = −459 mV vs. SHE, Ka,1 = 10 −6 (pKa,1 = 6), Ka,2 = 10 −8 (pKa,2 = 8), and a pH of 7 (A) or an applied potential of −400 mV vs. SHE (B). These values are arbitrary, but were chosen here to give conditions where significant PCET can be observed with an apparent redox midpoint potential of −400 mV and an apparent pKa value of 7.…”

“…However, it can be seen that Ox titrates to OxH and Red titrated to RedH as the pH is changed from high to low. 7, (11), (12), and 15, with = −400 mV, Ka,1 = Ka,2 = 10 −7 (pKa,1 = pKa,2 = 7), a pH of 7 (A) or an applied potential of −400 mV vs. SHE (B). These values are arbitrary, but were chosen here to give conditions where no PCET can be observed but conserve the redox midpoint potential of −400 mV and apparent pKa value of 7.…”

“…Variation of the fraction (out of 1) of the total oxidised (Ox + OxH, dark blue/red), the deprotonated oxidised (Ox, dashed blue/red), and the protonated oxidised (OxH, light blue/red) species as a function of the applied potential at pH 6 (red) and pH 8 (blue). The curves were obtained using Equations 7, (11), (12), and 15, with = −459 mV, Ka,1 = 10 −6 (pKa,1 = 6), and Ka,2 = 10 −8 (pKa,2 = 8).…”

“…For a long time, the major drawback of [FeFe] hydrogenases was their oxygen sensitivity [8][9][10]. However, methods that have recently been identified can protect these enzymes for later use [11][12][13][14], and even enzymes with a self-protection mechanism have been discovered [15,16].…”

Integrated bio-metal science: New frontiers of bio-metal science opened with cutting-edge techniques

“…NRVS is a unique technique using high intensity X-ray, produced by synchrotron radiation, to investigate the atomic vibration in molecules. Vibration modes of a specific atom, selectively excited via the nuclear level, give quite different and complementary information from that taken by Raman or infrared (IR) spectroscopy in the examination of the active center of metalloenzyme (Figure 4) [8][9][10][11][12]. X-rays from the synchrotron radiation can be also utilized for light source of new imaging systems that visualize biometal distribution and chemical status in tissues or even in cells.…”