Characterisation of the redox centers of ethylbenzene dehydrogenase.

Ethylbenzene dehydrogenase (EbDH), the initial enzyme of anaerobic ethylbenzene degradation from the beta-proteobacterium Aromatoleum aromaticum, is a soluble periplasmic molybdenum enzyme consisting of three subunits. It contains a Mo-bis-molybdopterin guanine dinucleotide (Mo-bis-MGD) cofactor and an 4Fe-4S cluster (FS0) in the α-subunit, three 4Fe-4S clusters (FS1 to FS3) and a 3Fe-4S cluster (FS4) in the β-subunit and a heme b cofactor in the γ-subunit. Ethylbenzene is hydroxylated by a water molecule in an oxygen-independent manner at the Mo-bis-MGD cofactor, which is reduced from the Mo to the Mo state in two subsequent one-electron steps. The electrons are then transferred via the Fe-S clusters to the heme b cofactor. In this report, we determine the midpoint redox potentials of the Mo-bis-MGD cofactor and FS1-FS4 by EPR spectroscopy, and that of the heme b cofactor by electrochemically induced redox difference spectroscopy. We obtained relatively high values of > 250 mV both for the Mo-Mo redox couple and the heme b cofactor, whereas FS2 is only reduced at a very low redox potential, causing magnetic coupling with the neighboring FS1 and FS3. We compare the results with the data on related enzymes and interpret their significance for the function of EbDH.