Direct Hemin Transfer from IsdA to IsdC in the Iron-regulated Surface Determinant (Isd) Heme Acquisition System of Staphylococcus aureus

Mengyao Liu,Wesley N Tanaka,Hui Zhu,Gang Xie,David M Dooley,Benfang Lei

doi:10.1074/jbc.m708372200

Mengyao Liu, Wesley N Tanaka + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m708372200

Copy DOI

Abstract

The iron-regulated surface determinants (Isd) of Staphylococcus aureus, including surface proteins IsdA, IsdB, IsdC, and IsdH and ATP-binding cassette transporter IsdDEF, constitute the machinery for acquiring heme as a preferred iron source. Here we report hemin transfer from hemin-containing IsdA (holo-IsdA) to hemin-free IsdC (apo-IsdC). The reaction has an equilibrium constant of 10 +/- 5 at 22 degrees C in favor of holo-IsdC formation. During the reaction, holo-IsdA binds to apo-IsdC and then transfers the cofactor to apo-IsdC with a rate constant of 54.3 +/- 1.8 s(-1) at 25 degrees C. The transfer rate is >70,000 times greater than the rate of simple hemin dissociation from holo-IsdA into solvent (k transfer = 54.3 s(-1) versus k -hemin = 0.00076 s(-1)). The standard free energy change, Delta G 0, is -27 kJ/mol for the formation of the holo-IsdA-apo-IsdC complex. IsdC has a higher affinity for hemin than IsdA. These results indicate that the IsdA-to-IsdC hemin transfer is through the activated holo-IsdA-apo-IsdC complex and is driven by the higher affinity of apo-IsdC for the cofactor. These findings demonstrate for the first time in the Isd system that heme transfer is rapid, direct, and affinity-driven from IsdA to IsdC. These results also provide the first example of heme transfer from one surface protein to another surface protein in Gram-positive bacteria and, perhaps most importantly, indicate that the mechanism of activated heme transfer, which we previously demonstrated between the streptococcal proteins Shp and HtsA, may apply in general to all bacterial heme transport systems.

Highlights

Cell surface heme-binding proteins have been identified in S. pyogenes [10], S. aureus [11], S. equi [9], and Bacillus anthracis [12], suggesting that, besides ATP-binding cassette (ABC) transporters, cell surface heme-binding proteins are required for heme acquisition by Gram-positive bacteria
The S. pyogenes system has a homologue in S. equi [9], whereas the homologue of the S. aureus system is present in B. anthracis [12]
Little is known about the biochemical mechanism of the heme acquisition by the iron-regulated surface determinants (Isd) system

Summary

EXPERIMENTAL PROCEDURES

Gene Cloning—The isdA and isdC genes were PCR-cloned from S. aureus subsp. aureus MW2 using paired primers 5ЈTACCATGGACAGCCAACAAGTCAATGCG-3Ј/5Ј-TGAATTCTTAAGTTTTTGGTAATTGTTTAGC-3Ј and 5Ј-ACCATGGATAGCGGTACTTTGAATTATG-3Ј/5Ј-AGAATTCTTATGTTTGTGGATTTTCTACTTTGTC-3Ј, respectively. Preparation of Holo-IsdA and Holo-IsdC—Holo-IsdC was obtained by reconstitution of its apo-form with hemin. One ml of apo-IsdC was incubated with excess hemin, loaded onto a Sephadex G-25 column (0.5 ϫ 30 cm), and eluted with 10 mM Tris-HCl, pH 8.0. Hemin Transfer—Holo-IsdA and apo-IsdC or holo-IsdC and apo-IsdA at indicated concentrations were incubated in 0.1 ml of 20 mM Tris-HCl, pH 8.0, at room temperature (22 °C) for 2 or 20 min. The concentrations of apo-IsdA and apo-IsdC in the same samples were calculated from A280 after subtracting the contribution from the holo-form using the extinction coefficients of 1.6 ϫ 104 and 1.8 ϫ 104 MϪ11⁄7cmϪ1, respectively. Kinetics of Hemin Transfer from Holo-IsdA to Apo-IsdC— The spectral changes associated with hemin transfer from holoIsdA to apo-IsdC were used to measure the transfer rate using the stopped-flow spectrophotometer at the indicated temperatures.

RESULTS

Kinetic parameter

DISCUSSION

Hemophore HasA