Investigations of two bidirectional carbon monoxide dehydrogenases from Carboxydothermus hydrogenoformans by protein film electrochemistry

Abstract

Carbon monoxide dehydrogenases (CODHs) catalyse the reversible conversion between CO and CO2 . Several small molecules or ions are inhibitors and probes for different oxidation states of the unusual [Ni-4 Fe-4 S] cluster that forms the active site. The actions of these small probes on two enzymes-CODH ICh and CODH IICh -produced by Carboxydothermus hydrogenoformans have been studied by protein film voltammetry to compare their behaviour and to establish general characteristics. Whereas CODH ICh is, so far, the better studied of the two isozymes in terms of its electrocatalytic properties, it is CODH IICh that has been characterised by X-ray crystallography. The two isozymes, which share 58.3% sequence identity and 73.9% sequence similarity, show similar patterns of behaviour with regard to selective inhibition of CO2 reduction by CO (product) and cyanate, potent and selective inhibition of CO oxidation by cyanide, and the action of sulfide, which promotes oxidative inactivation of the enzyme. For both isozymes, rates of binding of substrate analogues CN(-) (for CO) and NCO(-) (for CO2 ) are orders of magnitude lower than turnover, a feature that is clearly revealed through hysteresis of cyclic voltammetry. Inhibition by CN(-) and CO is much stronger for CODH IICh than for CODH ICh, a property that has relevance for applying these enzymes as model catalysts in solar-driven CO2 reduction.

Keywords: CO2 reduction; carbon monoxide dehydrogenases; cyclic voltammetry; inhibitors; protein film electrochemistry.

Figure 1

The overall structure of CODH II_Ch and different structures of the active sites (C-cluster): (a) −600 mV with CO₂, (b) CO-reduced CODH II_Ch, (c) −320 mV with cyanide. The dangling Fe-atom in the active site is found in two positions, labeled Fe_1a and Fe_1b, respectively. The PDB codes are shown in each case.

Figure 2

Voltammograms of CODH I_Ch and CODH II_Ch under 50% CO and 50% CO₂. Conditions: 25 °C, 0.2 M MES buffer (pH=7.0), rotation rate, 3500 rpm and scan rate, 2 mV sec⁻¹

Figure 3

Inhibition of CODH I_Ch and CODH II_Ch by cyanide under 100 % CO. An aliquot of KCN stock solution (giving a final concentration of 1mM in the electrochemical cell) was injected during Scan 2. Conditions: 25°C, 0.2 M MES buffer (pH=7.0), rotation rate, 3500 rpm and scan rate, 1mV sec⁻¹.

Figure 4

Inhibition of CODH I_Ch and CODH II_Ch by cyanide under 100% CO_2. An aliquot of KCN stock solution (giving a final concentration of 1mM in the electrochemical cell) was injected during Scan 2. Note that a more negative potential (by approximately 70 mV) is required to reactivate CODH II_Ch. Conditions: 25 °C, 0.2 M MES buffer (pH=7.0), rotation rate, 3500 rpm and scan rate, 1mV sec⁻¹.

Figure 5

Chronoamperometric measurements of the inactivation (Figure 5A and B) and re-activation (Figure 5C and D) rate of cyanide-inhibited CODH I_Ch and CODH II_Ch. The inactivation rate of CODH II_Ch by cyanide was measured at −460mV (CO oxidation, Figure 5A) and −560mV (CO₂ reduction, Figure 5B). A final concentration of 0.5 mM cyanide in the electrochemical cell was used to measure the half-life time for inactivation. Cyanide release from CODH II_Ch (Figure 5D) at −760mV is much faster than the instrumental response. Conditions: 25 °C, 0.2 M MES buffer (pH=7.0), and rotation rate, 3500 rpm.

Figure 6

Inhibition of CODH II_Ch by cyanate (8 mM final concentration). An aliquot of KOCN stock solution was injected into the cell at −760 mV at the beginning of Scan 2 under 20% CO and 80% CO_2. Conditions: 25 °C, 0.2 M MES buffer (pH=7.0), rotation rate, 3500 rpm and scan rate, 2mV sec⁻¹.

Figure 7

Investigations of the rate of inactivation of CODH I_Ch and CODH II_Ch by cyanate at −760 mV under different CO₂ concentrations (10% CO₂ or 100% CO₂). The normalized current is shown in figures and the fit to a single exponential decay curve is represented in the light grey line. Injections of KOCN were made into the electrochemical cell to give final concentrations of 6.7 mM for CODH I_Ch (upper figure) and 10mM for CODH II_Ch (lower figure). Conditions: 25°C, 0.2 M MES buffer (pH=7.0), and rotation rate, 3500 rpm.

Figure 8

Inhibition of CODH II_Ch by sulfide. An aliquot of sodium sulfide stock solution (giving 1mM final concentration) was injected into the electrochemical cell during Scan 2. The asterisk indicates that the re-activation potential occurs at −260 mV, which is more negative that observed for C_ox formed in the absence of sulfide. Conditions: 25°C, 0.2 M MES buffer (pH 7.0), rotation rate, 3500 rpm and scan rate, 1mV sec⁻¹.

Scheme 1

The catalytic cycle of CODH I_Ch and different catalytic states inhibited by small molecules. C_s is the sulfide-inhibited state.