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Journal of Bacteriology, October 2001, p. 5599-5608, Vol. 183, No. 19
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.19.5599-5608.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Binding Specificity of the Porphyromonas gingivalis Heme and Hemoglobin Receptor HmuR, Gingipain K, and Gingipain R1 for Heme, Porphyrins, and Metalloporphyrins

Teresa Olczak,1 Dabney White Dixon,2 and Caroline Attardo Genco1,*

Department of Medicine, Section of Infectious Diseases, Boston University School of Medicine, Boston, Massachusetts,1 and Department of Chemistry and Center for Biotechnology and Drug Design, Georgia State University, Atlanta, Georgia2

Received 2 March 2001/Accepted 5 July 2001

Previous genetic and biochemical studies have confirmed that hemoglobin and hemin utilization in Porphyromonas gingivalis is mediated by the outer membrane hemoglobin and heme receptor HmuR, as well as gingipain K (Kgp), a lysine-specific cysteine protease, and gingipain R1 (HRgpA), one of two arginine-specific cysteine proteases. In this study we report on the binding specificity of the recombinant P. gingivalis HmuR protein and native gingipains for hemoglobin, hemin, various porphyrins, and metalloporphyrins as assessed by spectrophotometric assays, by affinity chromatography, and by enzyme-linked immunosorbent assay. Protoporphyrin, mesoporphyrin, deuteroporphyrin, hematoporphyrin, and some of their iron, copper, and zinc derivatives were examined to evaluate the role of both the central metal ion and the peripheral substituents on binding to recombinant HmuR and soluble gingipains. Scatchard analysis of hemin binding to Escherichia coli cells expressing recombinant membrane-associated six-His-tagged HmuR yielded a linear plot with a binding affinity of 2.4 × 10-5 M. Recombinant E. coli cells bound the iron, copper, and zinc derivatives of protoporphyrin IX (PPIX) with similar affinities, and approximately four times more tightly than PPIX itself, which suggests that the active site of HmuR contains a histidine that binds the metal ion in the porphyrin ring. Furthermore, we found that recombinant HmuR prefers the ethyl and vinyl side chains of the PPIX molecule to either the larger hydroxyethyl or smaller hydrogen side chains. Kgp and HRgpA were demonstrated to bind various porphyrins and metalloporphyrins with affinities similar to those for hemin, indicating that the binding of Kgp and HRgpA to these porphyrins does not require a metal within the porphyrin ring. We did not detect the binding of RgpB, the arginine-specific cysteine protease that lacks a C-terminal hemagglutinin domain, to hemoglobin, porphyrins, or metalloporphyrins. Kgp and HRgpA, but not RgpB, were demonstrated to bind directly to soluble recombinant six-His-tagged HmuR. Several possible mechanisms for the cooperation between outer membrane receptor HmuR and proteases Kgp and HRgpA in hemin and hemoglobin binding and utilization are discussed.

* Corresponding author. Mailing address: Department of Medicine, Section of Infectious Diseases, Boston University School of Medicine, 650 Albany St., EBRC Bldg., Boston, MA 02118. Phone: (617) 414-5305. Fax: (617) 414-5280. E-mail: caroline.genco{at}bmc.org.

Journal of Bacteriology, October 2001, p. 5599-5608, Vol. 183, No. 19
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.19.5599-5608.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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