| Abstract
| - Carbon monoxide binding and inhibition have been investigated by electron paramagneticresonance (EPR) spectroscopy in solution and in crystals of structurally described states of the Fe-onlyhydrogenase (CpI) from Clostridiumpasteurianum. Simulation of the EPR spectrum of the as-isolatedstate indicates that the main component of the EPR spectrum consists of the oxidized state of the “Hcluster” and components due to reduced accessory FeS clusters. Addition of carbon monoxide to CpI inthe presence of dithionite results in the inhibition of hydrogen evolution activity, and a characteristicaxial EPR signal [geff(1), geff(2), and geff(3) = 2.0725, 2.0061, and 2.0061, respectively] was observed.Hydrogen evolution activity was restored by successive sparging with hydrogen and argon and resultedin samples that exhibited the native oxidized EPR signature that could be converted to the reduced formupon addition of sodium dithionite and hydrogen. To examine the relationship between the spectroscopicallydefined states of CpI and those observed structurally by X-ray crystallography, we have examined theCpI crystals using EPR spectroscopy. EPR spectra of the crystals in the CO-bound state exhibit thepreviously described axial signal associated with CO binding. The results indicate that the addition ofcarbon monoxide to CpI results in a single reversible carbon monoxide-bound species characterized byloss of enzyme activity and the distinctive axial EPR signal.
|
