MICROSCOPIC PATHWAY FOR THE MEDIUM-CHAIN FATTY ACYL COA DEHYDROGENASECATALYZED OXIDATIVE HALF-REACTION - CHANGES IN THE ELECTRONIC-STRUCTURES OF FLAVIN AND COA DERIVATIVES DURING CATALYSIS

In a previous communication, we demonstrated that the medium-chain fat ty acyl CoA dehydrogenase (MCAD) catalyzed conversion of 3-indolepropi onyl CoA (IPCoA) to trans-3-indoleacryloyl CoA (IACoA) proceeds via th e formation of an intermediary species X that possesses the electronic properties of reduced flavin and highly conjugated CoA product. Since the steady-state turnover of the enzyme-catalyzed dehydrogenation rea ction precisely matches with the rate of formation of X [Johnson, J. K ., & Srivastava, D. K. (1993) Biochemistry 32, 8004-8013], the latter species appeared to be the likely site for the transfer of electrons t o external electron acceptors (e.g., ferricenium hexafluorophosphate, FcPF6). To probe the microscopic pathway for the oxidative half-reacti on, we employed a sequential mixing stopped-flow technique utilizing I PCoA as the enzyme substrate and FcPF6 as the electron acceptor. The t ime-dependent changes in absorption at 450, 415, and 367 nm were measu red upon mixing FcPF6 with previously mixed and aged solutions of MCAD -FAD + IPCoA in the stopped-flow syringes. The kinetic traces show an increase (1/tau1) followed by a decrease (1/tau2) in absorption at 450 and 415 nm, and a lag (corresponding to the time regime of 1/tau1) fo llowed by an increase in absorption (1/tau2) at 367 nm. The relaxation rate constants (1/tau's) thus measured remain unaffected, with variat ions in the aging time; however, the amplitudes of these phases increa se up to the aging time of 5 s, after which the amplitudes attain maxi ma. For an aging time of 5 s, 1/tau1 and 1/tau2 show a linear and a hy perbolic dependence on the FcPF6 concentration, respectively. These, c oupled with the complementary studies involving butyryl CoA as a nonch romophoric substrate for this enzyme, lead us to propose the following sequence of events during the MCAD-catalyzed oxidative half-reaction: (1) The enzyme-catalyzed oxidative half-reaction proceeds via the for mation of a collision complex between X and FcPF6 during the fast (1/t au1) relaxation phase. (2) The reduced flavin moiety of X is oxidized via (rapid) transfer of electrons to FcPF6 within the collision comple x, without formation of a detectable (metastable) flavin semiquinone i ntermediate. (3) The transfer of electrons is accompanied by changes i n the electronic structures of both the flavin and IACoA moieties with in the enzyme-IACoA complex. The electronic structure of this newly fo rmed complex is exactly the same as that formed upon isomerization of the MCAD-FAD-IACoA complex [Johnson, J. K., Wang, Z. X., & Srivastava, D. K. (1992) Biochemistry 31, 10564-10575]. (4) The electronically pe rturbed flavin and IACoA species revert back to their normal structure s during the slow (1/tau2) relaxation phase. This step is followed by the dissociation of IACoA from the enzyme site to promote the subseque nt round of catalytic cycle.