Abstract
A heme domain of coral allene oxide synthase (cAOS) catalyzes the formation of allene oxide from fatty acid hydroperoxide. Although cAOS has a similar heme active site to that of catalase, cAOS is completely lacking in catalase activity. A close look at the hydrogen-bonding possibilities around the distal His in cAOS suggested that the imidazole ring is rotated by 180 degrees relative to that of catalase because of the hydrogen bond between Thr-66 and the distal His-67. This could contribute to the functional differences between cAOS and catalase, and to examine this possibility, we mutated Thr-66 in cAOS to Val, the corresponding residue in catalase. In contrast to the complete absence of catalase activity in wild type (WT) cAOS, T66V had a modest catalase activity. On the other hand, the mutation suppressed the native enzymatic activity of the formation of allene oxide to 14% of that of WT cAOS. In the resonance Raman spectrum, whereas WT cAOS has only a 6-coordinate/high spin heme, T66V has a 5-coordinate/high spin heme as a minor species. Because catalase adopts a 5-coordinate/high spin structure, probably the 5-coordinate/high spin portion of T66V showed the catalase activity. Furthermore, in accord with the fact that the CN affinity of catalase is higher than that of WT cAOS, the CN affinity of T66V was 8-fold higher than that of WT cAOS, indicating that the mutation could mimic the heme active site in catalase. We, therefore, propose that the hydrogen bond between Thr-66 and distal His-67 could modulate the orientation of distal His, thereby regulating the enzymatic activity in cAOS.
Highlights
A heme domain of coral allene oxide synthase catalyzes the formation of allene oxide from fatty acid hydroperoxide
CAOS cannot catalyze the dismutation of H2O2 to water and oxygen molecule, namely the catalase reaction (Scheme 2) [7, 9], whereas coral allene oxide synthase (cAOS), like bovine liver catalase (BLC), reacts with peracetic acid to form a ferryl oxo species with a tyrosine radical followed by the formation of a ferryl oxo porphyrin -cation radical species [10, 11]
Catalase Activity of the T66V cAOS Mutant—To examine the catalase activity of the T66V mutant, we evaluated the H2O2 consumption rate by monitoring the absorbance change of H2O2 after mixing 1 M T66V mutant and 10 mM H2O2
Summary
A heme domain of coral allene oxide synthase (cAOS) catalyzes the formation of allene oxide from fatty acid hydroperoxide. The conversion from fatty acid hydroperoxide to allene oxide in plants is catalyzed by a heme containing enzyme called allene oxide synthase (AOS) [2, 3]. As expected from the sequence homology, the spectroscopic data indicated that the heme active site structure in cAOS is quite similar to that of catalase [11, 12]. As compared with catalase, the recent crystal structure of cAOS showed the lack of hydrogen-bonding network involving the proximal tyrosinate ligand and the remarkable planarity of the heme [12].
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