Roles of P. gingivalis gingipains in haem pigment formation

Objectives: The black pigment of P. gingivalis, which is composed of the µ-oxo haem dimer, [Fe(III)]PPIX2O, is formed through degradation of both oxyhaemoglobin and deoxyhaemoglobin. Although a combination of both R- and K-gingipains is needed by cells to generate this haem species from oxyhaemoglobin (oxyHb) (Smalley et al., 2004, Biochem. J. 379, 833), the exact mechanisms are not clear. To determine the steps involved in [Fe(III)]PPIX]2O from oxyHb, interactions of oxyHb with isolated polyprotein R- and K- gingipains, HRgpA and Kgp, respectively, were studied. Methods: Soluble HRgpA and Kgp polyproteins were purified from the growth medium of strain HG66. UV-visible spectroscopy and SDS-PAGE was used to monitor the breakdown of horse oxyHb at pH 7.5 in 0.14M NaCl/0.1M Tris-HCl, pH 7.5, at 37°C. OxyHb and enzyme concentrations were 4 and 0.2µM, respectively. Results: Incubation of oxyHb with HRgpA resulted in formation of methaemoglobin (metHb) the oxidised form of haemoglobin. This step was prevented by the R-gingipain specific inhibitor leupeptin. In contrast, incubation of oxyHb with Kgp resulted in generation of an iron(III) haemoglobin haemichrome. This was inhibited by the lysine-specific protease inhibitor Z-Phe-Lys-acyloxymethylketone (Z-FKck). Although oxyHb was minimally degraded by Kgp, metHb, formed by treatment of oxyHb with NaNO2 or by pre-incubation with HRgpA, was rapidly degraded resulting in haem loss from the protein. MetHb degradation by Kgp was also inhibited BY Z-FKck. Conclusions: Together these data show that R-gingipain activity is crucial for converting oxyHb into the metHb form which is thus rendered more susceptible to Kgp degradation resulting in the eventual release of iron(III) protoporphyrin IX and production of the µ-oxo haem dimer.