Аннотация
The stearoyl-ACP delta 9 desaturase from plants is a new example of
a growing number of proteins that contain oxo- or hydroxo-bridged
diiron clusters. On the basis of differences in primary sequence
motifs providing the cluster ligands and upon structural differences
elucidated by X-ray crystallography, we now propose that the presently
known, soluble diiron-oxo proteins can be grouped into two classes,
I and II. Class I contains hemerythrin, myohemerythrin, and, possibly,
purple acid phosphatase. Class II contains ribonucleotide reductases,
bacterial hydrocarbon hydroxylases (methane monooxygenase, toluene-4-monooxygenase,
and phenol hydroxylase), rubrerythrin, and stearoyl-ACP desaturases.
Through the use of resonance Raman spectroscopy, we have detected
symmetric (vs = 519 cm-1) and asymmetric (vas = 747 cm-1) vibrational
modes in the castor stearoyl-ACP delta 9 desaturase, which are typical
of oxo-bridged diiron clusters. These frequencies shift by -18 and
-34 cm-1, respectively, in H218O, proving that the bridging ligand
is readily exchangeable with solvent (t1/2 = 7 min). Calculation
of an approximately 123 degrees Fe-O-Fe angle from the position
of vs and vas and from the 18O-dependent shift in these frequencies
suggests that the diiron-oxo cluster in the desaturase is triply
bridged in the diferric state. In the diferrous state, the two iron
sites of the cluster are structurally inequivalent, as shown by
differential temperature dependence of the M��ssbauer quadrupole
splittings. For the class II diiron-oxo proteins, primary sequence
alignments reveal conserved amino acid residues which act as iron
cluster ligands, participate in a hydrogen-bonding network, and
are potentially involved in O2 binding and activation. Based on
this conservation, a structural model for the stearoyl-ACP delta
9 desaturase active site is proposed that has strong similarity
to both ribonucleotide reductase and methane monooxygenase. However,
after single turnover of the diferous state with 18O2, 18O is not
detected in the oxo bridge of the castor desaturase. This is in
contrast to the outcome observed for ribonucleotide reductase, suggesting
the desaturase and ribonucleotide reductase differ in certain aspects
of their respective O2-activation reactions.
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