4-Hydroxybutyryl-coenzyme A (
CoA)
dehydratase (4HBD) from Clostridium aminobutyricum catalyzes the reversible
dehydration of
4-hydroxybutyryl-CoA to
crotonyl-CoA and the irreversible isomerization of
vinylacetyl-CoA to
crotonyl-CoA. 4HBD is an
oxygen-sensitive homotetrameric
enzyme with one [4Fe-4S](2+) cluster and one
flavin adenine dinucleotide (
FAD) in each subunit. Upon the addition of
crotonyl-CoA or the analogues
butyryl-CoA,
acetyl-CoA,
and CoA, UV-visible light and electron paramagnetic resonance (EPR) spectroscopy revealed an internal one-electron transfer to
FAD and the [4Fe-4S](2+) cluster prior to hydration. We describe an active recombinant 4HBD and variants produced in Escherichia coli. The variants of the cluster
ligands (H292C [
histidine at position 292 is replaced by
cysteine], H292E, C99A, C103A, and C299A) had no measurable
dehydratase activity and were composed of monomers, dimers, and tetramers. Variants of other potential catalytic residues were composed only of tetramers and exhibited either no measurable (E257Q, E455Q, and Y296W) hydratase activity or <1% (Y296F and T190V)
dehydratase activity. The E455Q variant but not the Y296F or E257Q variant displayed the same spectral changes as the wild-type
enzyme after the addition of
crotonyl-CoA but at a much lower rate. The results suggest that upon the addition of a substrate, Y296 is deprotonated by E455 and reduces
FAD to FADH·, aided by protonation from E257 via T190. In contrast to FADH·, the tyrosyl radical could not be detected by EPR spectroscopy. FADH· appears to initiate the radical
dehydration via an allylic ketyl radical that was proposed 19 years ago. The mode of radical generation in 4HBD is without precedent in anaerobic radical chemistry. It differs largely from that in
enzymes, which use
coenzyme B12,
S-adenosylmethionine,
ATP-driven electron transfer, or
flavin-based electron bifurcation for this purpose.