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J. Biol. Chem., Vol. 283, Issue 42, 28125-28136, October 17, 2008

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Demonstration of the Iron-regulated Surface Determinant (Isd) Heme Transfer Pathway in Staphylococcus aureus^*

Naomi Muryoi, Michael T. Tiedemann, Mark Pluym, Johnson Cheung, David E. Heinrichs¹, and Martin J. Stillman²

From the Department of Microbiology and Immunology, The University of Western Ontario, London, Ontario N6A 5C1, Canada and the Department of Chemistry, The University of Western Ontario, London, Ontario N6A 5B7, Canada

In this study, we report experimental results that provide the first complete challenge of a proposed model for heme acquisition by Staphylococcus aureus via the Isd pathway first put forth by Mazmanian, S. K., Skaar, E. P., Gaspar, A. H., Humayun, M., Gornicki, P., Jelenska, J., Joachmiak, A., Missiakas, D. M., and Schneewind, O. (2003) Science 299, 906–909. The heme-binding NEAT domains of Isd proteins IsdA, IsdB (domain 2), IsdC, and HarA/IsdH (domain 3), and the heme-binding IsdE protein, were overexpressed and purified in apo (heme-free) form. Absorption and magnetic circular dichroism spectral data, together with electrospray ionization mass spectrometry were used to unambiguously identify that heme transfers from NEAT-A through NEAT-C to IsdE. Heme transfer was demonstrated to occur in a unidirectional fashion in the sequence NEAT-B2 NEAT-A NEAT-C IsdE or, alternatively, initiating from NEAT-H3 instead of NEAT-B2: NEAT-H3 NEAT-A NEAT-C IsdE. Under the conditions of our experiments, only NEAT-H3 and NEAT-B2 could transfer bidirectionally, which is in the reverse direction as well, and only with each other. Whereas apo-IsdE readily accepted heme from holo-NEAT-C, it would not accept heme from holo-NEAT-A. Heme transfer to IsdE requires the presence of holo-NEAT-C, in agreement with the proposal that IsdC serves as the central conduit of the heme transfer pathway. These experimental findings corroborate the heme transfer model first proposed by the Schneewind group. Our data show that heme transport from the wall-anchored IsdH/IsdB proteins proceeds directly to IsdE at the membrane and, for this to occur, we propose that specific protein-protein interactions must take place.

Received for publication, March 19, 2008 , and in revised form, July 29, 2008.

^* This work was supported by a Canadian Institutes of Health Research operating grant (to D. E. H.), operating and equipment grants from the Natural Sciences and Engineering Research Council (NSERC) of Canada (to M. J. S.), the NSERC Postgraduate Scholarship program (to M. P.), and the Ontario Graduate Scholarship program (to M. T. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence may be addressed: Dept. of Microbiology and Immunology, University of Western Ontario, London, Ontario N6A 5C1, Canada. E-mail: deh{at}uwo.ca. ²To whom correspondence may be addressed: Dept. of Chemistry, University of Western Ontario, London, Ontario, N6A 5B7 Canada. E-mail: martin.stillman{at}uwo.ca.

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