A triplet spin system (
S = 1) is detected by low-temperature electron paramagnetic resonance(EPR) spectroscopy in samples of diol de
hydrase and t
he functional adenosylcobalamin (AdoCbl) analogue5'-deoxy-3',4'-an
hydroadenosylcobalamin (
anAdoCbl). Different spectra are observed in t
he presence andabsence of t
he substrate (
R,
S)-1,2-propanediol. In bot
h cases, t
he spectra include a prominent
half-fieldtransition (
hars/Delta.gif" BORDER=0 >
MS = 2) t
hat is a
hallmark of strongly coupled triplet spin systems. T
he appearance of
59Co
hyperfine splitting in t
he EPR signals and t
he positions (
g values) of t
he signals in t
he spectra s
how t
hat
half of t
he triplet spin is contributed by t
he low-spin Co
2+ of cob(II)alamin. Line widt
h effects fromisotopic labeling (
13C and
2H) in t
he 5'-deoxy-3',4'-an
hydroribosyl ring demonstrate t
hat t
he ot
her
half oft
he spin triplet is from an allylic 5'-deoxy-3',4'-an
hydroadenosyl (an
hydroadenosyl) radical. T
he zero-field splitting (ZFS) tensors describing t
he magnetic dipole-dipole interactions of t
he component spinsof t
he triplets
have r
hombic symmetry because of electron spin delocalization wit
hin t
he organic radicalcomponent and t
he proximity of t
he radical to t
he low-spin Co
2+. T
he dipole-dipole interaction wasmodeled as a summation of point-dipole interactions involving t
he spin-bearing orbitals of t
hean
hydroadenosyl radical and cob(II)alamin. Geometries w
hic
h are consistent wit
h t
he ZFS tensors in t
hepresence and absence of t
he substrate position t
he 5'-carbon of t
he an
hydroadenosyl radical 3.5 and 4.1Å from Co
2+, respectively. Homolytic cleavage of t
he cobalt-carbon bond of t
he analogue in t
he absenceof t
he substrate indicates t
hat, in diol de
hydrase, binding of t
he coenzyme to t
he protein weakens t
hebond prior to binding of t
he substrate.