Evidence that the greening ligand in native butyryl-CoA dehydrogenase is a CoA persulfide.

105

Thia- and oxaoctanoyl-CoA derivatives (substituted at the C-3 and C-4 positions) have been synthesized to prove the reductive half-reaction in the medium-chain acyl-CoA dehydrogenase from pig kidney. 3-Thiaoctanoyl-CoA binds to this flavoenzyme, forming an intense, stable, long-wavelength band (at 804 nm; extinction coefficient = 8.7 mM-1 cm-1 at pH 7.6). The intensity of this band increases about 20% from pH 6.0 to pH 8.8. This long-wavelength species probably represents a charge-transfer complex between bound acyl enolate as the donor and oxidized flavin adenine dinucleotide as the acceptor. Thus, the enzyme catalyzes alpha-proton exchange, and no long-wavelength bands are seen with 3-thiaoctyl-CoA (where the carbonyl moiety is replaced by a methylene group). 3-Oxaoctanoyl-CoA binds comparatively weakly to the dehydrogenase, with a long-wavelength band at 780 nm which is both less intense and less stable than the corresponding thia analogue. These data suggest that the enzyme can accomplish alpha-proton abstraction from certain weakly acidic acyl-CoA derivatives, without concerted transfer of a hydride equivalent to the flavin. 4-Thiaoctanoyl-CoA is dehydrogenated in the standard assay 1.5-fold faster than octanoyl-CoA. Titrations of the medium-chain dehydrogenase with the 4-thia derivative resemble those obtained with octanoyl-CoA, except for the contribution of the strongly absorbing 4-thia-trans-2-octenoyl-CoA product. The corresponding 4-oxa analogue is a much poorer substrate (10% of the rate shown by octanoyl-CoA) but again effects substantially complete reduction of the flavin chromophore in the dehydrogenase.(ABSTRACT TRUNCATED AT 250 WORDS)

Section: Resultsmentioning

confidence: 91%

The reductive half-reaction in acyl-CoA dehydrogenase from pig kidney: studies with thiaoctanoyl-CoA and oxaoctanoyl-CoA analogues

Lau¹,

Brantley²,

Thorpe³

1988

105

“…Recently, the ligand responsible for the green color of butyryl-CoA dehydrogenase has been identified as a coenzyme A persulfide (Williamson et al, 1982a,b). The addition of mixtures of CoAand S°containing species to pig kidney general acyl-CoA dehydrogenase also led to the appearance of a long-wavelength band centered at 710 nm, and experiments using flavin analogues (Williamson et al, 1982a) and resonance Raman spectroscopy (Williamson et al, 1982b) suggested that this new band be ascribed to a charge-transfer complex between oxidized flavin as the acceptor and bound persulfide as the donor. Surprisingly, acyl-CoA oxidase also reacts with CoA/S2"/S°m ixtures, generating a 710-nm band (Figure 6).…”

Section: Resultsmentioning

confidence: 99%

Acyl-CoA oxidase from Candida tropicalis

Jiang¹,

Thorpe²

1983

Acyl coenzyme A oxidase (acyl-CoA oxidase) has been isolated in good yield from Candida tropicalis pK 233 grown on n-alkanes. Gel filtration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and measurement of flavin content suggest that the oxidase is an octamer of Mr 75 000 subunits each containing one flavin. The oxidase yields the red semiquinone form on dithionite or photochemical reduction, slowly forms an N-5 adduct with 0.16 M sulfite at pH 7.4, and is rapidly reduced by borohydride, forming the 3,4-dihydroflavin isomer. The red flavosemiquinone is only kinetically stabilized with respect to disproportionation in the free enzyme but is thermodynamically stabilized on binding enoyl-CoA derivatives. The enzyme is reduced by butyryl-, octanoyl-, and palmitoyl-CoA without formation of prominent long-wavelength bands. Acyl-CoA oxidase and the acyl-CoA dehydrogenases share many similarities in their interaction with CoA derivatives. For example, both enzymes stabilize the anionic radical on binding enoyl-CoA derivatives, both dehydrogenate 2-oxoheptadecyldethio-CoA but cannot utilize S-heptadecyl-CoA, both form long-wavelength bands with CoA persulfide species, and both enzymes are attacked by the suicide substrates 3,4-pentadienoyl-CoA and (methylene-cyclopropyl)acetyl-CoA at the flavin prosthetic group.

“…In addition to DCIP, the suppression of reactivity of the enzyme by bound ligands is also encountered with dithionite as a reductant of the oxidized dehydrogenase, e.g., with CoA thioesters (Beinert & Page, 1957), with CoA persulfide (Engel & Massey, 1971;Williamson et al, 1982), and with 2-octynoyl-CoA-treated enzyme (Freund et al, 1985). Dithionite has been described as an inner-sphere reductant (Wherland & Gray, 1977) and as such would require close approach for effective delivery of reducing equivalents to the flavin.…”

Section: Discussionmentioning

confidence: 99%

Alternate electron acceptors for medium-chain acyl-CoA dehydrogenase: use of ferricenium salts

Lehman¹,

Thorpe²

1990

Medium-chain acyl-CoA dehydrogenase reduced with octanoyl-CoA is reoxidized in two one-electron steps by two molecules of the physiological oxidant, electron transferring flavoprotein (ETF). The organometallic oxidant ferricenium hexafluorophosphate (Fc+PF6-) is an excellent alternative oxidant of the dehydrogenase and mimics a number of the features shown by ETF. Reoxidation of octanoyl-CoA-reduced enzyme (200 microM Fc+PF6- in 100 mM Hepes buffer, pH 7.6, 1 degree C) occurs in two one-electron steps with pseudo-first-order rate constants of 40 s-1 and about 200 s-1 for k1 and k2, respectively. The reaction is comparatively insensitive to ionic strength, and evidence of rate saturation is encountered at high ferricenium ion concentration. As observed with ETF, the free two-electron-reduced dehydrogenase is a much poorer kinetic reductant of Fc+PF6-, with rate constants of 3 s-1 and 0.3 s-1 (for k1 and k2, respectively) using 200 microM Fc+PF6-. In addition to the enoyl-CoA product formed during the dehydrogenation of octanoyl-CoA, binding a number of redox-inert acyl-CoA analogues (notably 3-thia- and 3-oxaoctanoyl-CoA) significantly accelerates electron transfer from the dehydrogenase to Fc+PF6-. Those ligands most effective at accelerating electron transfer favor deprotonation of reduced flavin species in the acyl-CoA dehydrogenase. Thus this rate enhancement may reflect the anticipated kinetic superiority of anionic flavin forms as reductants in outer-sphere electron-transfer processes. Evidence consistent with the presence of two distinct loci for redox communication with the bound flavin in the acyl-CoA dehydrogenase is presented.