Ruffling of Metalloporphyrins Bound to IsdG and IsdI, Two Heme-degrading Enzymes in Staphylococcus aureus

Woo Cheol Lee,Michelle L Reniere,Eric P Skaar,Michael E.P Murphy

doi:10.1074/jbc.m709486200

Woo Cheol Lee, Michelle L Reniere + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m709486200

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Abstract

IsdG and IsdI are paralogous proteins that are intracellular components of a complex heme uptake system in Staphylococcus aureus. IsdG and IsdI were shown previously to reductively degrade hemin. Crystal structures of the apoproteins show that these proteins belong to a newly identified heme degradation family distinct from canonical eukaryotic and prokaryotic heme oxygenases. Here we report the crystal structures of an inactive N7A variant of IsdG in complex with Fe(3+)-protoporphyrin IX (IsdG-hemin) and of IsdI in complex with cobalt protoporphyrin IX (IsdI-CoPPIX) to 1.8 A or better resolution. These structures show that the metalloporphyrins are buried into similar deep clefts such that the propionic acids form salt bridges to two Arg residues. His(77) (IsdG) or His(76) (IsdI), a critical residue required for activity, is coordinated to the Fe(3+) or Co(3+) atoms, respectively. The bound porphyrin rings form extensive steric interactions in the binding cleft such that the rings are highly distorted from the plane. This distortion is best described as ruffled and places the beta- and delta-meso carbons proximal to the distal oxygen-binding site. In the IsdG-hemin structure, Fe(3+) is pentacoordinate, and the distal side is occluded by the side chain of Ile(55). However, in the structure of IsdI-CoPPIX, the distal side of the CoPPIX accommodates a chloride ion in a cavity formed through a conformational change in Ile(55). The chloride ion participates in a hydrogen bond to the side chain amide of Asn(6). Together the structures suggest a reaction mechanism in which a reactive peroxide intermediate proceeds with nucleophilic oxidation at the beta- or delta-meso carbon of the hemin.

Highlights

Have received significant attention because of the requirement of iron for the growth of most organisms [4]
The most striking features of the IsdG-hemin and IsdI-CoPPIX crystal structures are the highly distorted porphyrin rings. This structural similarity suggests that the porphyrin ring distortions are not artifacts of either the N7A substitution in IsdGhemin or the presence of a nondegraded heme analog in IsdICoPPIX
The IsdI-CoPPIX structure shows that ring distortion alone is insufficient for reductive degradation

Summary

EXPERIMENTAL PROCEDURES

Degradation of Metalloporphyrins—IsdG and IsdI were expressed in Escherichia coli BL21 (DE3) and purified as described previously [16]. To measure the oxygen dependence of the heme degradation reaction, oxygen was removed from solutions of hemin-reconstituted IsdG and IsdI (20 mM Tris, pH 7.5) by purging with argon in cuvettes sealed with a septum. Argon-purged ascorbate was added using a syringe, and UV-visible spectra were collected. Protein Expression and Crystallization—IsdG-N7A and IsdI were expressed in E. coli BL21 (DE3) and purified as reported previously [17]. Hemin and CoPPIX (Sigma) were dissolved in DMSO (10 mg/ml). Metalloporphyrin complexes were prepared by adding ϳ2:1 in molar ratios of hemin and CoPPIX to IsdG-N7A and IsdI, respectively. The protein was concentrated by Amicon centrifugal filters (molecular weight cut-off 10,000, Millipore) to a final concentration of ϳ15 mg/ml, as determined by the Bradford method using bovine serum albumin as a standard (Sigma)

Data collection and refinement statistics for the IsdG and IsdI complexes

RESULTS

DISCUSSION