Alcohol Intermediate in the Reaction Catalyzed by PHM
A R T I C L E S
Scheme 1. (a) Peptidylglycine R-Amidating Monooxygenase (PAM) and (b) Dopamine ꢀ-Monooxygenase (DꢀM) reactions
and His242) and a methionine sulfur ligand (Met314) and is
directly involved in dioxygen activation and substrate oxidation.
The other copper atom, CuH, has three Nδ-histidine ligands
(His107, His108, and His172) and is involved in electron transfer.
The PAL domain, unique to the PAM system, is zinc- and
calcium-dependent and catalyzes the dealkylation of the carbino-
lamide to the corresponding amide and glyoxylate (Scheme
1a).13,14
intramolecular electron transfer from the other Cu atom in the
active site, CuH, yielding a CuII-O• radical. Radical recombina-
tion of the substrate and Cu-O radical species produces an inner-
sphere alcohol intermediate. Subsequent hydrolysis of the inner-
sphere alcohol generates the hydroxylated product.
A highly reduced copper-oxo species is postulated for
substrate CR-H bond cleavage in both ‘copper-oxo’ mecha-
Intriguingly, DꢀM, without a PAL-like partner, will catalyze
oxidative dealkylation similar to the sequential reactions
catalyzed by PHM and PAL in bifunctional PAM.15 We report
herein the PAL-independent oxidative dealkylation of imino-
oxyacetic acids to the corresponding oximes and glyoxylate by
PHM. For PHM and DꢀM, the dealkylation reactions result
solely from the oxidation chemistry of their monooxygenase
domains. This similarity in dealkylation chemistry provides a
novel framework to study the mechanism of PAM catalysis.15-17
The role of oxygen activation in PHM and DꢀM has been
the subject of much study.18-22 The presumed nucleophile
responsible for hydrogen abstraction is predominantly considered
to be an end-on/η1 copper-superoxo radical species.11,23 Both
η1 and η2 copper-superoxo species had been prepared in models,
although none exhibited the propensity for both H-abstraction
and oxidation chemistry.24-29 Recently, the Karlin group
prepared end-on/η1 CuII-superoxo model complexes able to
orchestrate both H-abstraction and oxidation chemistry.30-33
Characterization of CuII-superoxo species was a critical advance;
however, ambiguities remain as to the identity of the actual
oxidant species.30
(10) Kulathila, R.; Consalvo, A. P.; Fitzpatrick, P. F.; Freeman, J. C.;
Snyder, L. M.; Villafranca, J. J.; Merkler, D. J. Arch. Biochem.
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(11) Prigge, S. T.; Eipper, B. A.; Mains, R. E.; Amzel, L. M. Science 2004,
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L. M. Nat. Struct. Biol. 1999, 6, 976–983.
(13) Kolhekar, A. S.; Bell, J.; Shiozaki, E. N.; Jin, L.; Keutmann, H. T.;
Hand, T. A.; Mains, R. E.; Eipper, B. A. Biochemistry 2002, 41,
12384–12394.
(14) De, M.; Bell, J.; Blackburn, N. J.; Mains, R. E.; Eipper, B. A. J. Biol.
Chem. 2006, 281, 20873–20882.
(15) Padgette, S. R.; Wimalasena, K.; Herman, H. H.; Sirimanne, S. R.;
May, S. W. Biochemistry 1985, 24, 5826–5839.
(16) Katopodis, A. G.; May, S. W. Biochemistry 1990, 29, 4541–4548.
(17) Katopodis, A. G.; Ping, D.; May, S. W. Biochemistry 1990, 29, 6115–
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(18) Gherman, B. F.; Heppner, D. E.; Tolman, W. B.; Cramer, C. J. J. Biol.
Inorg. Chem. 2006, 11, 197–205.
(19) Chen, P.; Solomon, E. I. J. Am. Chem. Soc. 2004, 126, 4991–5000.
(20) Evans, J. P.; Ahn, K.; Klinman, J. P. J. Biol. Chem. 2003, 278, 49691–
49698.
(21) Itoh, S. Curr. Opin. Chem. Biol. 2006, 10, 115–122.
(22) Hatcher, L. Q.; Karlin, K. D. J. Biol. Inorg. Chem. 2004, 9, 669–683.
(23) Bollinger, J. M., Jr.; Krebs, C. Curr. Opin. Chem. Biol. 2007, 11,
151–158.
(24) Fujisawa, K.; Tanaka, M.; Moro-oka, Y.; Kitajima, N. J. Am. Chem.
Soc. 1994, 116, 12079–12080.
The two PHM mechanisms which involve a CuII-superoxo
nucleophile for substrate activation differ in the initial coordina-
tion geometry of the dioxygen species to copper (Scheme 2).
The ‘side-on/η2’ mechanism is supported by spectroscopic
evidence from side-on/η2 Cu/O species model studies and is
predicted to have an antiferromagnetically coupled singlet
ground state (see Scheme 3).25,34 When compared with the end-
on/η1 species, the side-on/η2 copperII-superoxo was calculated
to be thermodynamically preferred for H-transfer.19,35 Evidence
for the end-on/η1 species comes from the PHM crystal struc-
ture11 and the work of Blackburn et al.,36 suggesting that this
species is the nucleophile responsible for CR-H abstraction.
Both mechanisms include an end-on/η1 CuII-hydroperoxo species
following CR-H bond cleavage. The ‘side-on/η2’ mechanism
involves a direct hydroxylation of the CR-substrate radical,
through a ‘water-assisted’ radical recombination reaction result-
ing in the simultaneous reduction of the CuII-hydroperoxo and
CR-OH product release. Conversely, the CuII-hydroperoxo
species from the ‘end-on/η1’ mechanism is reduced via an
(25) Chen, P.; Root, D. E.; Campochiaro, C.; Fujisawa, K.; Solomon, E. I.
J. Am. Chem. Soc. 2003, 125, 466–474.
(26) Karlin, K. D.; Wei, N.; Jung, B.; Kaderli, S.; Zuberbuhler, A. D. J. Am.
Chem. Soc. 1991, 113, 5868–5870.
(27) Karlin, K. D.; Wei, N.; Jung, B.; Kaderli, S.; Niklaus, P.; Zuberbuhler,
A. D. J. Am. Chem. Soc. 1993, 115, 9506–9514.
(28) Karlin, K. D.; Kaderli, S.; Zuberbuhler, A. D. Acc. Chem. Res. 1997,
30, 139–147.
(29) Schatz, M.; Raab, V.; Foxon, S. P.; Brehm, G.; Schneider, S.; Reiher,
S.; Holthausen, M. C.; Sundermeyer, J.; Schindler, S. Angew. Chem.,
Int. Ed. 2004, 43, 4306–4363.
(30) Maiti, D.; Sarjeant, A. A.; Karlin, K. D. J. Am. Chem. Soc. 2007,
129, 6720–6721.
(31) Maiti, D.; Lucas, H. R.; Sarjeant, A. A.; Karlin, K. D. J. Am. Chem.
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Sarjeant, A. A.; Incarvito, C. D.; Rheingold, A. L.; Hodgson, K. O.;
Hedman, B.; Solomon, E. I.; Karlin, K. D. Inorg. Chem. 2007.
(33) Maiti, D.; Fry, H. C.; Woertink, J. S.; Vance, M. A.; Solomon, E. I.;
Karlin, K. D. J. Am. Chem. Soc. 2007, 129, 264–265.
(34) Chen, P.; Solomon, E. I. J. Inorg. Biochem. 2002, 88, 368–374.
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