Reversible carbon monoxide binding and inhibition at the active site of the Fe-only hydrogenase.

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.