title: Peroxidase activity and involvement in the oxidative stress response of roseobacter denitrificans truncated hemoglobin.
abstract: Roseobacter denitrificans is a member of the widespread marine Roseobacter genus. We report the first characterization of a truncated hemoglobin from R. denitrificans (Rd. trHb) that was purified in the heme-bound form from heterologous expression of the protein in Escherichia coli. Rd. trHb exhibits predominantly alpha-helical secondary structure and absorbs light at 412, 538 and 572 nm. The phylogenetic classification suggests that Rd. trHb falls into group II trHbs, whereas sequence alignments indicate that it shares certain important heme pocket residues with group I trHbs in addition to those of group II trHbs. The resonance Raman spectra indicate that the isolated Rd. trHb contains a ferric heme that is mostly 6-coordinate low-spin and that the heme of the ferrous form displays a mixture of 5- and 6-coordinate states. Two Fe-His stretching modes were detected, notably one at 248 cm-1, which has been reported in peroxidases and some flavohemoglobins that contain an Fe-His-Asp (or Glu) catalytic triad, but was never reported before in a trHb. We show that Rd. trHb exhibits a significant peroxidase activity with a (kcat/Km) value three orders of magnitude higher than that of bovine Hb and only one order lower than that of horseradish peroxidase. This enzymatic activity is pH-dependent with a pKa value ~6.8. Homology modeling suggests that residues known to be important for interactions with heme-bound ligands in group II trHbs from Mycobacterium tuberculosis and Bacillus subtilis are pointing toward to heme in Rd. trHb. Genomic organization and gene expression profiles imply possible functions for detoxification of reactive oxygen and nitrogen species in vivo. Altogether, Rd. trHb exhibits some distinctive features and appears equipped to help the bacterium to cope with reactive oxygen/nitrogen species and/or to operate redox biochemistry.
authors: Department of Chemistry and Biochemistry, Clark University, Worcester, MA, United States of America.
authors: Yaya
authors: Y
authors: Wang
authors: Department of Biochemistry, Microbiology and Bioinformatics and PROTEO, Laval University, Quebec, Canada.
authors: Xavier
authors: X
authors: Barbeau
authors: Department of Chemistry and Biochemistry, Clark University, Worcester, MA, United States of America.
authors: Astha
authors: A
authors: Bilimoria
authors: Department of Biochemistry, Microbiology and Bioinformatics and PROTEO, Laval University, Quebec, Canada.
authors: Patrick
authors: P
authors: Lagüe
authors: Department of Biochemistry, Microbiology and Bioinformatics and PROTEO, Laval University, Quebec, Canada.
authors: Manon
authors: M
authors: Couture
authors: Department of Chemistry and Biochemistry, Clark University, Worcester, MA, United States of America.
authors: Joseph Kuo-Hsiang
authors: JK
authors: Tang
mesh_terms: D001412:Bacillus subtilis / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D008958:Models, Molecular; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000502:physiology*; D009195:Peroxidase / Q000378:metabolism*; D011487:Protein Conformation; D041904:Roseobacter / Q000378:metabolism*; D054793:Truncated Hemoglobins / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D054793:Truncated Hemoglobins; D009195:Peroxidase