Journal of the American Chemical Society
COMMUNICATION
presence of a lysine near N5; it plays no role in oxygen activation.
Other structural features must be responsible. Mutagenesis
eliminated the quinone binding bucket as the site of the reaction
of O2. The hydrophilic pyrimidine binding site over the si-face of
the flavin, ringed by hydrogen-bonding side chains, does not appear
to be the site of the reaction of O2 because the reduced enzymeÀ
orotate complex reacts only about 10-fold slower than the free
enzyme (Table 1).26 The site of the reaction of O2 in DHODs
remains a mystery. Our results suggest that many mechanisms are
possible for oxygen activation by flavoproteins. This is consistent
with the variety of structures of flavoproteins that react with
oxygen.3 A small, reactive molecule like O2, which appeared after
the origin of life and flavoenzymes, would be expected to find
many sites to react.
(11) Leferink, N. G.; Fraaije, M. W.; Joosten, H. J.; Schaap, P. J.;
Mattevi, A.; van Berkel, W. J. J. Biol. Chem. 2009, 284, 4392–7.
(12) Wang, R.; Thorpe, C. Biochemistry 1991, 30, 7895–901.
(13) Roth, J. P.; Klinman, J. P. Proc. Natl. Acad. Sci. U.S.A. 2003, 100,
62–7.
(14) Roth, J. P.; Wincek, R.; Nodet, G.; Edmondson, D. E.; McIntire,
W. S.; Klinman, J. P. J. Am. Chem. Soc. 2004, 126, 15120–31.
(15) Ghanem, M.; Gadda, G. Biochemistry 2005, 44, 893–904.
(16) Wagner, M. A.; Trickey, P.; Chen, Z. W.; Mathews, F. S.; Jorns,
M. S. Biochemistry 2000, 39, 8813–24.
(17) Wagner, M. A.; Jorns, M. S. Biochemistry 2000, 39, 8825–9.
(18) Trickey, P.; Wagner, M. A.; Jorns, M. S.; Mathews, F. S.
Structure 1999, 7 (3), 331–45.
(19) Zhao, G.; Bruckner, R. C.; Jorns, M. S. Biochemistry 2008,
47, 9124–35.
(20) Kommoju, P. R.; Chen, Z. W.; Bruckner, R. C.; Mathews, F. S.;
Jorns, M. S. Biochemistry 2011, 50, 5521–34.
(21) Bruckner, R. C.; Winans, J.; Jorns, M. S. Biochemistry 2011,
50, 4949–62.
(22) Gerhardinger, C.; Taneda, S.; Marion, M. S.; Monnier, V. M.
J. Biol. Chem. 1994, 269, 27297–302.
’ ASSOCIATED CONTENT
S
Supporting Information. Materials and methods pp
b
S2ÀS5. This material is available free of charge via the Internet
(23) Collard, F.; Fagan, R. L.; Zhang, J.; Palfey, B. A.; Monnier, V. M.
Biochemistry 2011, 50, 7977–7986.
(24) Cytochrome c, which is rapidly reduced by superoxide, was
included in oxidation mixtures in order to determine the reduced oxygen
species produced by the flavoenzyme. No superoxide was detected.
(25) Bj€ornberg, O.; Rowland, P.; Larsen, S.; Jensen, K. F. Biochem-
istry 1997, 36, 16197–205.
(26) Palfey, B. A.; Bj€ornberg, O.; Jensen, K. F. Biochemistry 2001,
40, 4381–90.
(27) Nørager, S.; Jensen, K. F.; Bj€ornberg, O.; Larsen, S. Structure
’ AUTHOR INFORMATION
Corresponding Author
Present Addresses
§Department of Biochemistry, University of Iowa, 51 Newton Rd.,
Iowa City, IA 52242.
de Duve Institute, Universitꢀe Catholique de Louvain, 75-39,
Avenue Hippocrate, 1200 Brussels, Belgium.
2002, 10, 1211–23.
(28) Macheroux, P.; Massey, V.; Thiele, D. J.; Volokita, M. Biochemistry
1991, 30, 4612–9.
(29) Rowland, P.; Nielsen, F. S.; Jensen, K. F.; Larsen, S. Structure
1997, 5, 239–52.
’ ACKNOWLEDGMENT
This work was supported by the National Institutes of Health
Grant GM61087 to B.A.P. R.L.F was supported by NIGMS
Training Grant GM07767. C.A.M. was supported by a University
of Michigan Rackham Merit Fellowship and National Institutes
of Health Grant GM61087-08-S1. F.C. was supported by the
Juvenile Diabetes Foundation and the Belgian F.N.R.S. V.M.M.
was supported by the National Institutes of Health Grant
EY07099. We thank Maria Nelson for her work on creating and
purifying the His19Asn and Arg102Met E.coli DHOD mutant
enzymes.
’ REFERENCES
(1) Bruice, T. C. Isr. J. Chem. 1984, 24, 54–61.
(2) Massey, V. J. Biol. Chem. 1994, 269, 22459–62.
(3) Fagan, R. L.; Palfey, B. A., Flavin-Dependent Enzymes; Elsevier:
Oxford, U.K., 2010; Vol. 7.
(4) Palfey, B. A.; McDonald, C. A. Arch. Biochem. Biophys. 2010,
493, 26–36.
(5) Pennati, A.; Gadda, G. Biochemistry 2011, 50, 1–3.
(6) Mattevi, A. Trends Biochem. Sci. 2006, 31, 276–83.
(7) Coulombe, R.; Yue, K. Q.; Ghisla, S.; Vrielink, A. J. Biol. Chem.
2001, 276, 30435–41.
(8) Chen, L.; Lyubimov, A. Y.; Brammer, L.; Vrielink, A.; Sampson,
N. S. Biochemistry 2008, 47, 5368–77.
(9) Baron, R.; Riley, C.; Chenprakhon, P.; Thotsaporn, K.; Winter,
R. T.; Alfieri, A.; Forneris, F.; van Berkel, W. J.; Chaiyen, P.; Fraaije,
M. W.; Mattevi, A.; McCammon, J. A. Proc. Natl. Acad. Sci. U.S.A. 2009,
106, 10603–8.
(10) Saam, J.; Rosini, E.; Molla, G.; Schulten, K.; Pollegioni, L.;
Ghisla, S. J. Biol. Chem. 2010, 285, 24439–46.
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dx.doi.org/10.1021/ja2081873 |J. Am. Chem. Soc. 2011, 133, 16809–16811