Reaction of Mycobacterium tuberculosis truncated hemoglobin O with hydrogen peroxide: evidence for peroxidatic activity and formation of protein-based radicals.
In this work, we investigated the reaction of ferric Mycobacterium tuberculosis truncated hemoglobin O (trHbO) with hydrogen peroxide. Stopped-flow spectrophotometric experiments under single turnover conditions showed that trHbO reacts with H(2)O(2) to give transient intermediate(s), among which is an oxyferryl heme, different from a typical peroxidase Compound I (oxyferryl heme pi-cation radical). EPR spectroscopy indicated evidence for both tryptophanyl and tyrosyl radicals, whereas redox titrations demonstrated that the peroxide-treated protein product retains 2 oxidizing eq. We propose that Compound I formed transiently is reduced with concomitant oxidation of Trp(G8) to give the detected oxoferryl heme and a radical on Trp(G8) (detected by EPR of the trHbO Tyr(CD1)Phe mutant). In the wild-type protein, the Trp(G8) radical is in turn reduced rapidly by Tyr(CD1). In a second cycle, Trp(G8) may be reoxidized by the ferryl heme to yield ferric heme and two protein radicals. In turn, these migrate to form tyrosyl radicals on Tyr(55) and Tyr(115), which lead, in the absence of a reducing substrate, to oligomerization of the protein. Steady-state kinetics in the presence of H(2)O(2) and the one-electron donor 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) indicated that trHbO has peroxidase activity, in accord with the presence of typical peroxidase intermediates. These findings suggest an oxidation/reduction function for trHbO and, by analogy, for other Group II trHbs.
Ouellet
Hugues
H
Department of Biochemistry and Microbiology, Laval University, Quebec G1K 7P4, Canada.
Ranguelova
Kalina
K
Labarre
Marie
M
Wittenberg
Jonathan B
JB
Wittenberg
Beatrice A
BA
Magliozzo
Richard S
RS
Guertin
Michel
M
D001426:Bacterial Proteins; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals; D006454:Hemoglobins / Q000737:chemistry*; D006861:Hydrogen Peroxide / Q000737:chemistry*; D007700:Kinetics; D010544:Peroxidases / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D054793:Truncated Hemoglobins
D001426:Bacterial Proteins; D005609:Free Radicals; D006454:Hemoglobins; D054793:Truncated Hemoglobins; C451738:hemoglobin O, Mycobacterium tuberculosis; D006861:Hydrogen Peroxide; D010544:Peroxidases