Peroxidation and redox reactions catalyzed by truncated hemoglobins

Abstract

Roseobacter denitrificans is one member of widespread marine Rosoebacters and performs photosynthesis and denitrification aerobically. We report a native heme‐bound R. denitrificans truncated hemoglobin (Rd. trHb) from heterologous expression in E. coli. Rd. trHb exhibits absorbance at 412, 538 and 572 nm bands with predominately alpha‐helical secondary structure. The phylogenetic classification suggests that Rd. trHb falls into group II trHbs, whereas sequence alignments indicate that it shares important heme pocket residues with both groups I and II trHbs. The resonance Raman spectra indicate that ferric Rd. trHb is mostly 6‐coordinate low‐spin and the ferrous form is a mixture of 5‐ and 6‐coordinated states. Two Fe‐His stretching modes were detected: one at 228 cm‐1, which represents an unconstrained neutral histidine as in other trHbs, and the other at 248 cm‐1, which has been reported in peroxidases and some flavohemoglobins that contain Fe‐His‐Asp (or Glu) catalytic triad, but not in trHbs. Further, Rd. trHb exhibits a significant peroxidase activity with a (kcat/Km) value three orders of magnitude higher than bovine Hb and only one order lower than horseradish peroxidase (HRP). The pH‐rate profiles yield 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 M. tuberculosis and B. 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 distinct features and appears equipped to help Roseobacters to cope with reactive oxygen/nitrogen species and/or operate redox biochemistry.