Porphyrin-mediated binding to hemoglobin by the HA2 domain of cysteine proteinases (gingipains) and hemagglutinins from the periodontal pathogen Porphyromonas gingivalis

Heme binding and uptake are considered fundamental to the growth and virule nce of the gram-negative periodontal pathogen Porphyromonas gingivalis. We therefore examined the potential role of the dominant P. gingivalis cystein e proteinases (gingipains) in the acquisition of heme from the environment, A recombinant hemoglobin-binding domain that is conserved between two pred ominant gingipains (domain HA2) demonstrated tight binding to hemin (K-d = 16 nM), and binding was inhibited by iron-free protoporphyrin IX (K-i = 2.5 mu M). Hemoglobin binding to the gingipains and the recombinant HA2 (rHA2) domain (K-d = 2.1 nM) was also inhibited by protoporphyrin IX (K-i 10 mu M ), demonstrating an essential interaction between the HA2 domain and the he me moiety in hemoglobin binding. Binding of rHA2 with either hemin, protopo rphyrin IX, or hematoporphyrin was abolished by establishing covalent linka ge of the protoporphyrin propionic acid side chains to fixed amines, demons trating specific and directed binding: of rHA2 to these protoporphyrins. A monoclonal antibody which recognizes a peptide epitope within the HA2 domai n was employed to demonstrate that HA2-associated hemoglobin-binding activi ty was expressed and released by P. gingivalis cells in a batch culture, in parallel with proteinase activity. Cysteine proteinases from P. gingivalis appear to be multidomain proteins with functions for hemagglutination, ery throcyte lysis, proteolysis, and heme binding, as demonstrated here. Detail ed understanding of the biochemical pathways for heme acquisition in P. gin givalis may allow precise targeting of this critical metabolic aspect for p eriodontal disease prevention.