Ligand-Binding and Substrate Turnover of a Heme Peroxidase from the Diatom Thalassiosira Pseudonana

examining the chemical and biophysical properties of recombinant THB1 (rTHB1) produced in E. coli as an apoprotein and recombined with a b heme. The goal of the present work was to validate the use of rTHB1 as a surrogate for the native protein. THB1 was partially purified from whole cell extracts of C. reinhardtii by chromatographic methods. Enhanced chemiluminescence staining and immunodetection of the protein mixture after native gel electrophoresis showed that THB1 has peroxidase activity. Ultra performance liquid chromatography and mass spectrometry confirmed the association of the polypeptide with a b heme and revealed that THB1 is N-terminally acetylated. Nanodrop analysis of the purified extracts returned an optical spectrum consistent with that of recombinant ferric THB1. In addition, the mixture had nitric oxide dioxygenase activity, as observed for rTHB1 [1]. The combined information provides compelling evidence that THB1 uses a b heme as cofactor and that the properties of rTHB1 are relevant to THB1 as it is found within the living cell. The observation of a co-translational modification cautions against the sole use of sequence information to derive physiological insight. [1] Johnson et al. (2014) Biochemistry 53:4573 Supported by NSF grant MCB-1330488