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J Biol Chem, Vol. 275, Issue 12, 8582-8591, March 24, 2000
From the Department of Molecular Biology, Scripps Research
Institute, La Jolla, California 92037
Heme enzymes are capable of catalyzing a range of
oxidative chemistry with high specificity, depending on the surrounding protein environment. We describe here a reaction catalyzed by a mutant
of cytochrome c peroxidase, which is similar but distinct from those catalyzed by nitric-oxide synthase. In the R48A mutant, an
expanded water-filled cavity was created above the distal heme face.
N-Hydroxyguanidine (NHG) but not guanidine was shown to bind in the cavity with Kd = 8.5 mM,
and coordinate to the heme to give a low spin state. Reaction of R48A
with peroxide produced a Fe(IV)=O/Trp·+ center
capable of oxidizing either NHG or
N The atomic coordinates and structure factors (codes 1DJ1 and
1DJ5) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New
Brunswick, NJ (http://www.rcsb.org/).
Unusual Oxidative Chemistry of
N
-Hydroxyarginine and
N-Hydroxyguanidine Catalyzed at an Engineered Cavity in
a Heme Peroxidase*
and
-hydroxyarginine (NHA), but not arginine or
guanidine, by a multi-turnover catalytic process. Oxidation of either
NHG or NHA by R48A did not result in the accumulation of NO,
NO2
, NO3, urea, or citrulline, but
instead afforded a yellow product with absorption maxima of 257 and 400 nm. Mass spectrometry of the derivatized NHA products identified the
yellow species as N-nitrosoarginine. We suggest that a
nitrosylating agent, possibly derived from HNO, is produced by the
oxidation of one molecule of substrate. This then reacts with a second
substrate molecule to form the observed N-nitroso products.
This complex chemistry illustrates how the active sites of enzymes such
as nitric-oxide synthase may serve to prevent alternative reactions
from occurring, in addition to enabling those desired.
*
This work was supported by a Wellcome Trust Prize
International Research Fellowship to J. H and by National Institutes of Health Grant GM41049 (to D. B. G.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Supported by a Wellcome Trust Prize International Research
Fellowship. Current address: Medical Research Council, Dunn Human Nutrition Unit, Hills Rd. Cambridge, CB2 2XY, UK.
§
To whom correspondence should be addressed: Dept. of Molecular
Biology, MB8, Scripps Research Inst., 10550 N. Torrey Pines Rd., La
Jolla, CA 92037. Tel.: 858-784-9892; Fax: 858-784-2857; E-mail:
dbg@scripps.edu.
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