Abstract
Ethylbenzene dehydrogenase (EbDH), the initial enzyme of anaerobic ethylbenzene degradation from the beta-proteobacterium Aromatoleumaromaticum, 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 MoVI to the MoIV 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 MoVI–MoV 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.Graphical abstract
Highlights
Ethylbenzene dehydrogenase (EbDH) is the initial enzyme of anaerobic ethylbenzene degradation in the denitrifying beta-proteobacterium Aromatoleum aromaticum [1,2,3]
The enzyme stays in the oxidised form and is resistant to exposure to air, while reduced EbDH is readily inactivated by oxygen [4]
We investigate here the redox properties of the metal cofactors of EbDH, which mediate electron transfer through the enzyme from the molybdenum cofactor in the active site to the heme b as putative electron exit site
Summary
Ethylbenzene dehydrogenase (EbDH) is the initial enzyme of anaerobic ethylbenzene degradation in the denitrifying beta-proteobacterium Aromatoleum aromaticum [1,2,3]. The redox properties of the cofactors involved in electron transfer in enzymes of subfamily II have so far been reported for nitrate reductase [14, 26, 27], selenate reductase [28] and dimethylsulfide dehydrogenase [29, 30] All of these enzymes have in common a relatively low midpoint potential of the second [Fe4S4] cluster of the β-subunit, whereas the potentials of the other redox cofactors vary between the enzymes [14, 28, 30]. These data contribute to the further elucidation of the mechanisms of electron flow from the catalytically active Mo-cofactor to the heme b implied in electron exit. EbdA 73-KWDKVNWGSHL.NICWPQGSCKFYVYVRNGIVWREEQAAQTPACNVDYVDYNPLGCQKGSA pCyA 54-TWDKVVRSTHH.LNCWYQAHCSWDVYVKDGLVYREEQAGEYPQVNPQLPDFNPRGCQKGGC C25A 61-KWDRVVKGTHTRANCI..GACSWDVYVKDGIAWREEQAAIYEPHRPDIPDFNPRGCKGACY NarG_Ec 41-QHDKIVRSTHG.VNCT..GSCSWKIYVKNGLVTWETQQTDYPRTRPDLPNHEPRGCPRGAS Nar_Ha 70-DWDSVARSTHS.VNCT..GSCSWNVYVKDGQVWREEQAGDYPTFDESLPDPNPRGCQKGAC Nar_Ap 73-AYDKVARSTHG.VNCT..GSCSWMVYVKDGIVAYELQAGDYPDIGPSYPNYEPRGCPRGAS Nar_Pa 59-QYDKVARSTHG.VNCT..GSCSWNVYVKDGLIVWELQATDYPDISPDIPNYEPRGCPRGAS SerA 64-TWDSTGFITHS.NGCV..AGCAWRVFVKNGVPMREEQVSEYPQL.PGVPDMNPRGCQKGAV ClrA 60-TWDSVGVMTHS.NGCV..AGCAWNVFVKNGIPMREEQISKYPQL.PGIPDMNPRGCQKGAV DdhA 57-TWDYVGKAAHC.INCL..GNCAFDIYVKDGIVIREEQLAKYPQISPDIPDANPRGCQKGAI PcrA 51-SWDKKTRGAHL.INCT..GACPHFVYTKDGVVIREEQSKDIPPM.PNIPELNPRGCNKGEC
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