Histidine pK(a) shifts and changes of tautomeric states induced by the binding of gallium-protoporphyrin IX in the hemophore HasA(SM)

Abstract

The HasA(SM) hemophore, secreted by Serratia marcescens, binds free or hemoprotein bound heme with high affinity and delivers it to a specific outer membrane receptor, HasR. In HasA(SM), heme is held by two loops and coordinated to iron by two residues, His 32 and Tyr 75. A third residue His 83 was shown recently to play a crucial role in heme ligation. To address the mechanistic issues of the heme capture and release processes, the histidine protonation states were studied in both apo- and holo-forms of HasA(SM) in solution. Holo-HasA(SM) was formed with gallium-protoporphyrin IX (GaPPIX), giving rise to a diamagnetic protein. By use of heteronuclear correlation NMR spectroscopy, the imidazole side-chain (15)N and (1)H resonances of the six HasA(SM) histidines were assigned and their pKa values and predominant tautomeric states according to pH were determined. We show that protonation states of the heme pocket histidines can modulate the nucleophilic character of the two axial ligands and, consequently, control the heme binding. In particular, the essential role of the His 83 is emphasized according to its direct interaction with Tyr 75.

Fig. 1.

Portions of ¹H{¹⁵N}MBC spectra of uniformly ¹⁵N labeled HasA in the apo-form (left) and in the holo-form (right) in 20 mM sodium phosphate buffer (pH 5.6) and 30°C. Only His 17, His 32, His 83, His 128, and His 133 cross peak patterns are indicated.

Fig. 2.

Heme pocket histidine ¹⁵N chemical shifts of HasA_SM for the apo-form (left) and for the holo-form (right) as a function of pH. The solid lines represent the least-squares best fit to the data. The titration curve for the His 83 ¹H resonance in the apo-HasA_SM is shown in the bottom right corner because only partial ¹⁵N titration curves didn't allow to determine the pKa value by fitting the data for this residue.

Fig. 3.

(A) Ribbon representation of the overall HasA X-ray crystal structure with the location of b heme, histidines, and Tyr 75 residues (PDB entry 1B2V; Arnoux et al. 1999). (B) Detailed view of the b heme structural environment. Nitrogens are shown in green, oxygen in light blue, hydrogens in black, carbons in red, and backbone in yellow.

Fig. 4.

Schematic representation of the Tyr 75–His 83 interaction in apo-Has_SM (left) and in GAPPIX-Has_SM (right) at neutral pH. The dotted lines represent the hydrogen bond between the two residues.