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 (k cat/K m) 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 pK a 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.
Highlights
Marine Roseobacters [1,2,3] are known to be abundant in the ocean ecosystem near the surface [4], and recent studies indicate that they are responsible for producing reactive oxygenPLOS ONE | DOI:10.1371/journal.pone.0117768 February 6, 2015Peroxidase Activity by Roseobacters hemoglobin
The glbO gene encoding truncated hemoglobins (trHbs) from R. denitrificans (Rd. trHb) was sub-cloned and the recombinant protein was heterologously expressed in E. coli and purified using the protocol reported for a group II trHb from Mycobacterium tuberculosis (Mt. trHbO) [52] with minor modifications
We have isolated a trHb from R. denitrificans cultures
Summary
Marine Roseobacters [1,2,3] are known to be abundant in the ocean ecosystem near the surface [4], and recent studies indicate that they are responsible for producing reactive oxygen. Roseobacter denitrificans, which may form a symbiotic relationship with marine eukaryotic communities as it was isolated from seaweeds in Tokyo Bay [6], is an aerobic anoxygenic (non-oxygen evolving) photosynthetic bacterium. It is one of the first Roseobacters being characterized [7] and the first Roseobacter species whose genome was sequenced [8]. Optical absorption spectra of R. denitrificans cultures display wavelength maxima at 808 and 873 nm (light-harvesting antenna complexes), 450–600 nm (photosynthetic pigments) and ~400 nm The latter indicates the presence of various hemoproteins, such as cytochromes and hemoglobins (Hbs). Potential biological roles of Rd. trHb are proposed and the relation with the aerobic denitrification process by R. denitrificans is discussed
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