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 p aramagnetic resonance (EPR) spectroscopy in solution and in crystals of str ucturally described states of the Fe-only hydrogenase (CpI) from Clostridiu m pasteurianum. Simulation of the EPR spectrum of the as-isolated state ind icates that the main component of the EPR spectrum consists of the oxidized state of the "H cluster" and components due to reduced accessory FeS clust ers. Addition of carbon monoxide to CpI in the presence of dithionite resul ts in the inhibition of hydrogen evolution activity, and a characteristic a xial 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 restore d by successive sparging with hydrogen and argon and resulted in samples th at exhibited the native oxidized EPR signature that could be converted to t he reduced form upon addition of sodium dithionite and hydrogen. To examine the relationship between the spectroscopically defined states of CpI and t hose observed structurally by X-ray crystallography, we have examined the C pI crystals using EPR spectroscopy. EPR spectra of the crystals in the GO-b ound 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.