Acyl-CoA dehydrogenases constitute a family of flavoproteins that catalyze the a,b-dehydrogenation of fatty acid acyl-CoA conjugates. While they differ widely in their specificity, they share the same basic chemical mechanism of a,b-dehydrogenation. Medium chain acyl-CoA dehydrogenase is probably the best-studied member of the class and serves as a model for the study of catalytic mechanisms. Based on medium chain acyl-CoA dehydrogenase we discuss the main factors that bring about catalysis, promote specificity and determine the selective transfer of electrons to electron transferring flavoprotein. The mechanism of a,bdehydrogenation is viewed as a process in which the substrate aC-H and bC-H bonds are ruptured concertedly, the first hydrogen being removed by the active center base Glu376-COO -as an H + , the second being transferred as a hydride to the flavin N(5) position. Hereby the pK a of the substrate aC-H is lowered from > 20 to % 8 by the effect of specific hydrogen bonds. Concomitantly, the pK a of Glu376-COO -is also raised to 8-9 due to the decrease in polarity brought about by substrate binding. The kinetic sequence of medium chain acyl-CoA dehydrogenase is rather complex and involves several intermediates. A prominent one is the molecular complex of reduced enzyme with the enoyl-CoA product that is characterized by an intense charge transfer absorption and serves as the point of transfer of electrons to the electron transferring flavoprotein. These views are also discussed in the context of the accompanying paper on the three-dimensional properties of acyl-CoA dehydrogenases.

Ero1p is a key enzyme in the disulfide bond formation pathway in eukaryotic cells in both aerobic and anaerobic environments. It was previously demonstrated that Ero1p can transfer electrons from thiol substrates to molecular oxygen. However, the fate of electrons under anaerobic conditions and the final fate of electrons under aerobic conditions remained obscure. To address these fundamental issues in the Ero1p mechanism, we studied the transfer of electrons from recombinant yeast Ero1p to various electron acceptors. Under aerobic conditions, reduction of molecular oxygen by Ero1p yielded stoichiometric hydrogen peroxide. Remarkably, we found that reduced Ero1p can transfer electrons to a variety of small and macromolecular electron acceptors in addition to molecular oxygen. In particular, Ero1p can catalyze reduction of exogenous FAD in solution. Free FAD is not required for the catalysis of dithiol oxidation by Ero1p, but it is sufficient to drive disulfide bond formation under anaerobic conditions. These findings provide insight into mechanisms for regenerating oxidized Ero1p and maintaining disulfide bond formation under anaerobic conditions in the endoplasmic reticulum.electron transfer ͉ Ero1 ͉ flavoenzyme ͉ hydrogen peroxide T he thiol oxidase enzyme Ero1p (1, 2), conserved across eukaryotes, generates disulfide bonds de novo in the endoplasmic reticulum (ER) by catalyzing the transfer of electrons from dithiols to molecular oxygen (3). The Ero1p active site consists of a Cys-Xaa-Xaa-Cys amino acid sequence motif (in which Xaa is a non-Cys amino acid) juxtaposed with the isoalloxazine ring system of a bound FAD cofactor (4) (Fig. 1). An additional redox center, a Cys-Xaa 4 -Cys disulfide, has been proposed to accept electrons from substrate proteins and transfer them to the Cys-Xaa-Xaa-Cys disulfide (4, 5).The arrangement of bound flavin, fixed active-site disulfide, and flexible shuttle disulfide is also found in Erv2p (6), a second yeast ER thiol oxidase with no sequence similarity to Ero1p (7,8). Erv2p is a member of the QSOX͞ALR enzyme family (9). The overall reaction of the QSOX͞ALR enzymes can be divided into two half-reactions. In the reductive half-reaction, the enzyme accepts electrons from reducing substrates, resulting in a reduction of the bound flavin cofactor. In the oxidative halfreaction, the enzyme deposits the electrons on an acceptor, such as molecular oxygen, to restore the bound cofactor to its initial state, as shown in Eqs. 1 and 2.Although this scheme describes the simplest scenario, four-, six-, and even eight-electron-reduced states of these f lavindependent sulfhydryl oxidase enzymes may also be possible, depending on the number of redox-active Cys pairs and the rate of internal equilibration between them (10, 11).It is likely that the basic reaction scheme described above for the QSOX͞ALR family applies to Ero1p as well. First, the similar arrangements of the functional groups in the active sites of Ero1p and Erv2p (4) suggest that the two thiol oxidase families may share feat...

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