Structures of the Heme Acquisition Protein HasA with Iron(III)‐5,15‐Diphenylporphyrin and Derivatives Thereof as an Artificial Prosthetic Group

Abstract

Iron(III)-5,15-diphenylporphyrin and several derivatives were accommodated by HasA, a heme acquisition protein secreted by Pseudomonas aeruginosa, despite possessing bulky substituents at the meso position of the porphyrin. Crystal structure analysis revealed that the two phenyl groups at the meso positions of porphyrin extend outside HasA. It was shown that the growth of P. aeruginosa was inhibited in the presence of HasA coordinating the synthetic porphyrins under iron-limiting conditions, and that the structure of the synthetic porphyrins greatly affects the inhibition efficiency.

Tetraphenylporphyrin Enters the Ring: First Example of a Complex Between Highly Bulky Porphyrins and a Protein

“…Growth experiments of P. aeruginosa were performed according to a previously reported method. 35 1 µM holo-HasA was added as the sole iron source to all cultures. To evaluate the efficiency of growth inhibition of HasA capturing TPP derivatives, these artificial-HasA were also added to a final concentration of 1 µM.…”

“…1a), can accept various bulky synthetic metalloporphyrins, such as diphenylporphyrin. [34][35][36][37] Yet, at this time, we were unable to incorporate bulkier TPP into HasA and concluded that this may not be possible, 34,35 and that the TPP skeleton may be incompatible for incorporation into unadulterated proteins and application with natural biological systems.…”

“…Our research group has previously reported that HasA capturing iron phthalocyanine and iron 5,15-diazaporphyrin inhibit the proliferation of P. aeruginosa under iron-limiting condition. 34,35 Initial investigation was conducted on a standard strain of P. aeruginosa called PAO1. In iron-limiting growth medium, P. aeruginosa PAO1 grew through acquisition of haem from holo-HasA that had been supplemented to the medium as the sole source of iron.…”

Highly malleable haem-binding site of the haemoprotein HasA permits stable accommodation of bulky tetraphenylporphycenes

“…Expression and purication of truncated HasA derived from P. aeruginosa PAO1 was performed according to similar methods reported previously. 13,14 The details are given in the ESI. † The purity of the protein was checked by SDS-PAGE.…”

“…Recently, we reported that the haem acquisition protein HasA secreted by Pseudomonas aeruginosa is rather promiscuous and can accommodate other metal complexes, such as insoluble iron(III)-phthalocyanine, small iron(III)-salophen, and bulky iron(III)-5,15-diphenylporphyrin. 13,14 HasA is a haem-binding protein that is secreted by certain pathogens, such as P. aeruginosa, when in iron decient environments. Following acquisition of haem from its host by HasA, haem is transferred to the HasA-specic outer membrane receptor HasR, where it is taken up into the cell and used as an iron source for the pathogen's survival ( Fig.…”