Using a functional enzyme model to understand the chemistry behind hydrogen sulfide induced hibernation

Abstract

The toxic gas H(2)S is produced by enzymes in the body. At moderate concentrations, H(2)S elicits physiological effects similar to hibernation. Herein, we describe experiments that imply that the phenomenon probably results from reversible inhibition of the enzyme cytochrome c oxidase (CcO), which reduces oxygen during respiration. A functional model of the oxygen-reducing site in CcO was used to explore the effects of H(2)S during respiration. Spectroscopic analyses showed that the model binds two molecules of H2S. The electro-catalytic reduction of oxygen is reversibly inhibited by H(2)S concentrations similar to those that induce hibernation. This phenomenon derives from a weak, reversible binding of H(2)S to the Fe(II) porphyrin, which mimics heme a(3) in CcO's active site. No inhibition of CcO is detected at lower H(2)S concentrations. Nevertheless, at lower concentrations, H(2)S could have other biological effects on CcO. For example, H(2)S rapidly reduces Fe(III) and Cu(II) in both the oxidized form of this functional model and in CcO itself. H(2)S also reduces CcO's biological reductant, cytochrome c, which normally derives its reducing equivalents from food metabolism. Consequently, it is speculated that H(2)S might also serve as a source of electrons during periods of hibernation when food supplies are low.

Fig. 1.

Active site structures. CcO (A) and synthetic model (B) of CcO (Fe Cu and phenol analogue).

Fig. 2.

Schematic representation of the FeCuPhOH catalyst “clicked” onto an Au electrode (white) with Pt ring (gray) around it. “Cat” refers to the catalyst (Fig. 1B), excluding the terminal alkyne moiety.

Fig. 3.

The electrocatalytic reduction of oxygen is reversibly inhibited by H2S. (A) A linear sweep voltammogram of the FeCuPhOH catalyst modified electrode showing catalytic O₂ reduction current under fast electron flux in the absence of NaSH (black), in the presence of 68 μM NaSH (red), 134 μM NaSH (blue), after removal of NaSH (dotted green), and in 200 μM NaSH in absence of any catalyst (dotted red). (B) Plot of percent catalytic current vs. NaSH concentration. (C) Linear sweep voltammogram of the FeCuPhOH catalyst modified electrode showing catalytic O₂ reduction current under slow (rate determining) electron flux in the absence of NaSH (black), in the presence of 50 μM NaSH (red), 100 μM NaSH (blue), 200 μM NaSH (green, and afterremoval of NaSH (dotted green) .

Fig. 4.

¹H-NMR spectrum (recorded in CD₂Cl₂ at −50 °C, 300 MHz) of Fe^II complexes (14 mM) before A and after B exposure to H₂S.

Fig. 5.

Infrared spectrum of the H₂S- and D₂S-complexes (in black and red, respectively) on KBr pellets or in solution in dichloromethane in a KBr cell (10 mM).

Fig. 6.

UV/Vis spectrum of a 18 μM Fe(II) complex before reaction with H₂S (dry, 5-coordinate, 430 nm in black) and after reaction (6-coordinate, 427 nm in red).

Fig. 7.

MSMS analysis on the most aboundant isotopic peak in the high-resolution nano-spray mass spectrum of the Fe(II) H₂S (A, Calcd: 1414.3771, Found: 1414.3793) and D₂S complexes (B, Calcd: 1420.4148, Found: 1420.3739), respectively. For this peak

Fig. 8.

The reactivity of Fe(II) H2S complexes with CO and O2 monitored by UV-Vis spectroscopy. (A) UV–Vis spectra of reduced 5 μM Fe^IICu^IPhOH (black), reduced Fe^IICu^IPhOH + 0.5 mM NaSH solution (red), and reduced Fe^IIPhOH + 0.5 mM NaSH solution + excess CO gas (blue). (B) Reduced 8 μM Fe^IIPhOH (black), reduced Fe^IIPhOH + 80 μM NaSH solution (red), and reduced Fe^IIPhOH + 80 μM NaSH solution + excess O₂ gas (green).

Fig. 9.

The reactivity of fully oxidized complexes with H2S monitored by UV-Vis and EPR spectroscopies. (A) UV–Vis spectra (in a 50:50 volume ratio of pH 7 aqueous buffer and acetonitrile) of 10 μM oxidized Fe^IIICu^IIPhOH (red), oxidized Fe^IIICu^IIPhOH + 70 μM NaSH solution (blue) overlaid with a reduced Fe^IICu^IPhOH spectrum (dotted black). (B) EPR spectra of 1 mM oxidized Fe^IIICu^II (red) in THF and oxidized Fe^IIICu^II + 1 mM (t-Bu₄N)SH solution (blue) at 77 K.

Scheme 1.

Schematic representation of the possible roles of H₂S affecting CcO activity. The blue box indicates that H₂S binds to the reduced CcO active site, but can be subsequently replaced by O₂. This inhibition should occur at moderate H₂S concentrations. At lower concentrations, H₂S does not compete with the substrate O₂ but can still reduce CcO's active site and/or cytochrome c (Cytc) during catalytic O₂ reduction.