title: A combination of both arginine- and lysine-specific gingipain activity of Porphyromonas gingivalis is necessary for the generation of the micro-oxo bishaem-containing pigment from haemoglobin.
abstract: The black pigment of Porphyromonas gingivalis is composed of the mu-oxo bishaem complex of Fe(III) protoporphyrin IX (mu-oxo oligomer, dimeric haem), namely [Fe(III)PPIX]2O. P. gingivalis W50 and Rgp (Arg-gingipain)- and Kgp (Lys-gingipain)-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 the production of mu-oxo bishaem. Mu-oxo Bishaem was detected by UV-visible, Mössbauer and Raman spectroscopies in wild-type W50 and in the black-pigmented RgpA- and RgpB-deficient mutants (W501 and D7 respectively), whereas no haem species were detected in the straw-coloured colonies of Kgp-deficient strain K1A. The dark brown pigment of the double RgpA/RgpB knockout mutant (E8) was not composed of mu-oxo bishaem, but of a high-spin monomeric Fe(III) protoporphyrin IX species (possibly a haem-albumin complex). In vitro incubation of oxyhaemoglobin with cells of the W50 strain and the RgpA- and RgpB-deficient mutants (W501 and D7) resulted in the formation of mu-oxo bishaem via methaemoglobin as an intermediate. Although the Kgp-deficient strain K1A converted oxyhaemoglobin into methaemoglobin, this was not further degraded into mu-oxo bishaem. The double RgpA/RgpB knockout was also not capable of producing mu-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 the production of mu-oxo bishaem from oxyhaemoglobin, but resulted in the formation of a haemoglobin haemichrome. These results show that one or both of RgpA and RgpB gingipains, in addition to the lysine-specific gingipain, is necessary for the production of mu-oxo bishaem from haemoglobin by whole cells of P. gingivalis.
authors.affiliation: Unit of Basic Dental Science, Department of Clinical Dental Sciences, University of Liverpool, The Edwards Building, Daulby Street, Liverpool L69 3GN, UK. josmall@liv.ac.uk
authors.forename: John W
authors.initials: JW
authors.lastname: Smalley
authors.forename: Michael F
authors.initials: MF
authors.lastname: Thomas
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Birss
authors.forename: Robert
authors.initials: R
authors.lastname: Withnall
authors.forename: Jack
authors.initials: J
authors.lastname: Silver
mesh_terms: D018829:Adhesins, Bacterial; D000362:Agar; D000818:Animals; D003546:Cysteine Endopeptidases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D000080867:Gingipain Cysteine Endopeptidases; D006388:Hemagglutinins / Q000235:genetics / Q000378:metabolism*; D006736:Horses / Q000097:blood; D007976:Leupeptins / Q000378:metabolism; D010108:Oxyhemoglobins / Q000378:metabolism*; D010860:Pigments, Biological / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D016966:Porphyromonas gingivalis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D011524:Protoporphyrins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D015204:Spectroscopy, Mossbauer; D013059:Spectrum Analysis, Raman; D013997:Time Factors
chemical_list: D018829:Adhesins, Bacterial; D000080867:Gingipain Cysteine Endopeptidases; D006388:Hemagglutinins; D007976:Leupeptins; D010108:Oxyhemoglobins; D010860:Pigments, Biological; D011524:Protoporphyrins; C489143:mu-oxo dimeric Fe(III)-protoporphyrin IX; D000362:Agar; D003546:Cysteine Endopeptidases; C032854:leupeptin