Monooxygenation of an aromatic ring by F43W/H64D/V68I myoglobin mutant and hydrogen peroxide. Myoglobin mutants as a model for P450 hydroxylation chemistry.
Myoglobin (Mb) is used as a model system for other heme proteins and the reactions they catalyze. The latest novel function to be proposed for myoglobin is a P450 type hydroxylation activity of aromatic carbons (Watanabe, Y., and Ueno, T. (2003) Bull. Chem. Soc. Jpn. 76, 1309-1322). Because Mb does not contain a specific substrate binding site for aromatic compounds near the heme, an engineered tryptophan in the heme pocket was used to model P450 hydroxylation of aromatic compounds. The monooxygenation product was not previously isolated because of rapid subsequent oxidation steps (Hara, I., Ueno, T., Ozaki, S., Itoh, S., Lee, K., Ueyama, N., and Watanabe, Y. (2001) J. Biol. Chem. 276, 36067-36070). In this work, a Mb variant (F43W/H64D/V68I) is used to characterize the monooxygenated intermediate. A modified (+16 Da) species forms upon the addition of 1 eq of H2O2. This product was digested with chymotrypsin, and the modified peptide fragments were isolated and characterized as 6-hydroxytryptophan using matrix-assisted laser desorption ionization time-of-flight tandem mass spectroscopy and 1H NMR. This engineered Mb variant represents the first enzyme to preferentially hydroxylate the indole side chain of Trp at the C6 position. Finally, heme extraction was used to demonstrate that both the formation of the 6-hydroxytryptophan intermediate (+16 Da) and subsequent oxidation to form the +30 Da final product are catalyzed by the heme cofactor, most probably via the compound I intermediate. These results provide insight into the mechanism of hydroxylation of aromatic carbons by heme proteins, demonstrating that non-thiolate-ligated heme enzymes can perform this function. This establishes Mb compound I as a model for P450 type aromatic hydroxylation chemistry.
Pfister
Thomas D
TD
Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, 61820, USA.
Ohki
Takahiro
T
Ueno
Takafumi
T
Hara
Isao
I
Adachi
Seiji
S
Makino
Yumiko
Y
Ueyama
Norikazu
N
Lu
Yi
Y
Watanabe
Yoshihito
Y
D006916:5-Hydroxytryptophan / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002374:Catalase / Q000737:chemistry; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000737:chemistry; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D006418:Heme / Q000737:chemistry; D006420:Hemeproteins; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008954:Models, Biological; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation; D009211:Myoglobin / Q000737:chemistry / Q000235:genetics*; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry; D017680:Potassium Compounds / Q000737:chemistry; D017434:Protein Structure, Tertiary; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013053:Spectrophotometry; D014364:Tryptophan / Q000737:chemistry; D014466:Ultraviolet Rays; D014907:Whales
D006420:Hemeproteins; D009211:Myoglobin; D010455:Peptides; D010710:Phosphates; D017680:Potassium Compounds; D006418:Heme; D014364:Tryptophan; D003577:Cytochrome P-450 Enzyme System; C013216:potassium phosphate; D006861:Hydrogen Peroxide; D006916:5-Hydroxytryptophan; D002374:Catalase; D002918:Chymotrypsin; D010100:Oxygen