Catabolism of nonphenolic arenes is frequently initiated by dioxygenases, yielding single isomer products with two adjacent hydroxylated asymmetric centers. The next enzymic reaction dehydrogenates these cyclic cis-diols, with aromatization yielding catechols for ring cleavage. H was transferred from both the 2-and 3-C atoms of (2S,3R)-p-toluate-2,3-dihydrodiol with specifically deuterated species in approximately equal amounts; and (iii) the unexpected lack of stereo-and regioselectivity of p-toluate-2,3-diol dehydrogenase was supported by kinetic isotope effect studies.Most nonhydroxylated arenes biodegraded by bacteria are dihydroxylated initially, in NADH-dependent reactions, to give cis-1,2-diol cyclohexadiene intermediates (10,(12)(13)(14)17). The second reaction of these catabolic pathways is usually an NADdependent dehydrogenation with concomitant rearomatization of the ring to give a catechol. The seminal work of Gibson (11) established this central, and apparently sole, preserve of bacterial evolution; cis-1,2-diol formation in other biological kingdoms has not yet been described.These dihydroxylation and dehydrogenation systems pose several questions of enzyme stereospecificity. Two asymmetric centers are formed in a single initial reaction, and they are destroyed in the next catabolic reaction by dehydrogenation to catechols. There are therefore two possible stereochemical questions to address for each reaction, to describe the diastereochemistry of these enzymic reactions. (i) Which hydrogen of NADH (4R or 4S) is transferred for the dihydroxylation? (ii) Which atom in the diol product receives hydrogen (H ϩ or H Ϫ )? For the successive dehydrogenation reactions, the inverse questions require solution. (iii) To which face of the nicotinamide ring of NAD ϩ is hydride ion transferred? (iv) From which C center of the dihydrodiol is hydride transferred to give NADH. In this paper we provide answers to questions ii, iii, and iv.Previously we have shown that the flavoprotein hydroxylases for orcinol, resorcinol, and 3-hydroxybenzoate (4-and 6-hydroxylases) donate the (4S) proton from NADH, by 3 H and 2 H isotope experiments (30, 37). You et al. (38) confirmed these results by proton nuclear magnetic resonance (NMR) experiments with five enzymes that we provided. A recent study by Schläfli et al. (34), using the tritium method to discriminate the (4S) and (4R) protons in NADH, examined the stereospecificity of hydride (tritide) transfer by the flavin cytochrome reductases of several arene-cis-1,2-dihydroxylases. There appears to be a consistency, since the examined enzymes of class I, e.g., phthalate dioxygenase and 2-chlorobenzoate dioxygenase, used the (4S)-3
