Reversible Carbon Monoxide Binding and Inhibition at the Active Site of the Fe-Only Hydrogenase

Abstract

Carbon monoxide binding and inhibition have been investigated by electron paramagnetic resonance (EPR) spectroscopy in solution and in crystals of structurally described states of the Fe-only hydrogenase (CpI) from Clostridium pasteurianum. Simulation of the EPR spectrum of the as-isolated state indicates that the main component of the EPR spectrum consists of the oxidized state of the "H cluster" and components due to reduced accessory FeS clusters. Addition of carbon monoxide to CpI in the presence of dithionite results in the inhibition of hydrogen evolution activity, and a characteristic axial EPR signal [g(eff(1)), g(eff(2)), and g(eff(3)) = 2.0725, 2.0061, and 2.0061, respectively] was observed. Hydrogen evolution activity was restored by successive sparging with hydrogen and argon and resulted in samples that exhibited the native oxidized EPR signature that could be converted to the reduced form upon addition of sodium dithionite and hydrogen. To examine the relationship between the spectroscopically defined states of CpI and those observed structurally by X-ray crystallography, we have examined the CpI crystals using EPR spectroscopy. EPR spectra of the crystals in the CO-bound state exhibit the previously described axial signal associated with CO binding. The results indicate that the addition of carbon monoxide to CpI results in a single reversible carbon monoxide-bound species characterized by loss of enzyme activity and the distinctive axial EPR signal.

Carbon monoxide conversion by thermophilic sulfate-reducing bacteria in pure culture and in co-culture with Carboxydothermus hydrogenoformans

“…When the acetogenic bacterium Eubacterium limosum was grown with a mixture of CO and H 2 (Sharak Genthner and Bryant 1982), H 2 consumption started after the CO concentration in the gas phase had decreased to values below 5%. This observation was explained by the possible inhibition of hydrogenase by CO. A similar hydrogenase inhibition was found for other anaerobic bacteria cultivated on CO (Daniels et al 1977;Pankhania et al 1986;Berlier et al 1987;Adams 1990;Bennett et al 2000).…”

The organometallic active site of [Fe]hydrogenase: Models and entatic states

“…Thus as electrons enter or leave the dinuclear active-site cluster, the -CO is well situated to moderate charge differences with only minor structural changes. Interpretation of Mössbauer (44) and EPR data (45,46) and correlation of IR spectra with model compounds (40) (44)(45)(46). This bridging CO (Ϸ1,800 cm…”

Commercial Biomass Syngas Fermentation

“…This depends if CO serves as both carbon and energy source, or if an additional energy source such hydrogen is present which can be utilised in a hydrogenase reaction (as illustrated in Figure 1). It should be noted though that electron production is thermodynamically more favourable from CO than from H 2 [96], and hydrogenases are reversibly inhibited by CO [97][98][99].…”