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J. Biol. Chem., Vol. 279, Issue 28, 29805-29815, July 9, 2004

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Characterization of the Heme Environment in Arabidopsis thaliana Fatty Acid -Dioxygenase-1^*

Wen Liu, Corina E. Rogge, Bijan Bambai, Graham Palmer¶, Ah-Lim Tsai, and Richard J. Kulmacz||

From the Department of Internal Medicine, University of Texas Health Science Center, Houston, Texas 77030 and the ^¶Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005

Plant -dioxygenases (PADOX) are hemoproteins in the myeloperoxidase family. We have used a variety of spectroscopic, mutagenic, and kinetic approaches to characterize the heme environment in Arabidopsis thaliana PADOX-1. Recombinant PADOX-1 purified to homogeneity contained 1 mol of heme bound tightly but noncovalently per protein monomer. Electronic absorbance, electron paramagnetic resonance, and magnetic circular dichroism spectra showed a high spin ferric heme that could be reduced to the ferrous state by dithionite. Cyanide bound relatively weakly in the ferric PADOX-1 heme vicinity (K_d 10 mM) but did not shift the heme to the low spin state. Cyanide was a very strong inhibitor of the fatty acid oxygenase activity (K_i 5 µM) and increased the K_m value for oxygen but not that for fatty acid. Spectroscopic analyses indicated that carbon monoxide, azide, imidazole, and a variety of substituted imidazoles did not bind appreciably in the ferric PADOX-1 heme vicinity. Substitution of His-163 and His-389 with cysteine, glutamine, tyrosine, or methionine resulted in variable degrees of perturbation of the heme absorbance spectrum and oxygenase activity, consistent with His-389 serving as the proximal heme ligand and indicating that the heme has a functional role in catalysis. Overall, A. thaliana PADOX-1 resembles a b-type cytochrome, although with much more restricted access to the distal face of the heme than seen in most other myeloperoxidase family members, explaining the previously puzzling lack of peroxidase activity in the plant protein. PADOX-1 is unusual in that it has a high affinity, inhibitory cyanide-binding site distinct from the distal heme face and the fatty acid site.

Received for publication, January 20, 2004 , and in revised form, April 9, 2004.

^* This work was supported by National Institutes of Health Grants GM44911 (to A.-L. T.) and GM52170 (to R. J. K.), Postdoctoral Fellowship DK61929 (to C. E. R.), and by Welch Foundation Grant C-636 (to G. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: National Research Center for Genetic Engineering and Biotechnology, P. O. Box 14155-6343, Tehran, Iran.

^|| To whom correspondence should be addressed: Dept. of Internal Medicine, University of Texas Health Science Center, MSB 5.284, 6431 Fannin St., Houston, TX 77030. Tel.: 713-500-6772; Fax: 713-500-6810; E-mail: richard.j.kulmacz{at}uth.tmc.edu.

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