RoxP binds heme
It has long been recognized that P. acnes produces and secretes porphyrins20,26 as byproducts of the vitamin B12 biosynthesis pathway. The high production of porphyrins from P. acnes is exploited therapeutically to kill the bacterium27. Porphyrin-like molecules are usually used as co-factors in several proteins (such as the methylmalony-CoA mutase). It has not been studied if P. acnes secretes proteins containing porphyrins (e.g. protoporphyrin IX; further on denoted as heme). To investigate if RoxP could interact with heme, we studied its binding to hemin-agarose. RoxP bound strongly to the column (Fig. 3b), and could also be stained with the heme-specific stain o-dianisidine (Fig. 3c). Furthermore, incubation of RoxP with heme and subsequent desalting to remove excess of unbound heme revealed a protein with an absorbance spectrum characteristic for heme proteins with the absorbance peak around 400 nm (Soret band) (Fig. 3d).
RoxP acts as an antioxidant
Many heme proteins can modulate the redox status of molecules, being able to reduce oxidized macromolecules28. To investigate if RoxP has antioxidant effects, we incubated purified RoxP with preformed ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)-radicals. Addition of 0.34 μM RoxP was able to reduce the free radicals in a time-dependent manner, reaching a plateau after 12 min (Fig. 4a,b). Increasing concentrations of RoxP resulted in immediate reduction of the absorbance to background levels. PBS served as a negative control, and vitamin E as a positive internal control, with the latter demonstrating a similar degree of antioxidant activity as RoxP