A combination of both arginine- and lysine-specific gingipain activity of Porphyromonas gingivalis is necessary for the generation of the m-oxo bishaem-containing pigment from haemoglobin

Abstract

The black pigment of Porphyromonas gingivalis is composed of the μ-oxo bishaem complex of iron(III) protoporphyrin IX (μ-oxo oligomer, dimeric haem), [Fe(III)PPIX]2O. P. gingivalis W50 and Rgp- and Kgp-gingipain protease-deficient mutants K1A, D7, E8 and W501 (Aduse-Opoku, , Davies, Gallagher, Hashim, Evans, Rangarajan, Slaney and Curtis (2000) Microbiology 146 , 1933-1940) were grown on horse blood agar for 14 days and examined for production of μ-oxo bishaem. The μ-oxo bishaem was detected by UV-visible, Mössbauer and Raman spectroscopy in wild type W50, and in the black-pigmented RgpA-deficient and RgpB-deficient mutants (W501 and D7), whilst no haems were detected in the straw-coloured Kgp-deficient strain K1A. The dark brown pigment of the double RgpA/RgpB knockout mutant (E8) was not composed of μ-oxo bishaem, but of a high-spin monomeric iron(III) protoporphyrin IX species (possibly a haem-albumin complex). In vitro incubation of oxyhaemoglobin with cells of strains W50, and the RgpA- and RgpB-deficient mutants (W501 and D7) resulted in formation of μ-oxo bishaem via methaemoglobin as an intermediate. Whilst the Kgp-deficient strain K1A converted oxyhaemoglobin into methaemoglobin, this was not further degraded into μ-oxo bishaem. The double RgpA/RgpB knockout was also unable to produce μ-oxo bishaem from oxyhaemoglobin, but instead generated a haemoglobin haemichrome. Inhibition of Arg-X protease activity of W50, W501, D7 and K1A with leupeptin under conditions where Lys-X protease activity was unaffected, prevented μ-oxo bishaem production from oxyhaemoglobin, but resulted in formation of a haemoglobin haemichrome. These data show that one or both of RgpA- and RgpB-gingipains, in addition to the lysine-specific gingipain, is necessary for production of the μ-oxo bishaem from haemoglobin by whole cells of P. gingivalis.