ATP as an Allosteric Modulator and Chelator of Fe3+ from Fe3+‐transferrin. Is ATP a Major Fe3+ Carrier

Abstract

Listen

Translate article

The objectives of this study were to examine two chelators in the removal of Fe3+ from Fe3+‐transferrin. One was pyrophosphate (PPi) as it is a reasonably effective chelator in this reaction, and the other was ATP in that it consists of PPi attached to adenosine monophosphate. We reasoned that ATP might bind to a nucleotide binding site if present and in close proximity to the metal center, to enhance Fe3+ removal and give insight into the mechanism of Fe3+ release. In this study we used uv/vis spectroscopy to examine the removal of Fe3+ from Fe3+‐transferrin by time‐course and Vi reactions. At acidic pH we determined association constants of Fe3+‐transferrin alone, and Fe3+‐transferrin in the presence of ATP. We also employed molecular modeling to identify and characterize a putative ATP binding site. Our results show that sigmoidal kinetics were obtained as a function of pH when ATP was the chelator. At pH 7.5 the kinetic data showed there was no removal of Fe3+ by ATP. ATP was found to exert positive heterotropic effects at acidic pH and resulted in the increase in the association constant of Fe3+‐transferrin. These effects were not seen with PPi. We concluded that there were two destabilizations of Fe3+‐transferrin necessary for Fe3+ release; the first due to a low pH induced conformational change in the protein and the second due to ATP, also at low pH, where ATP served as an allosteric modulator. Essentially 100% of Fe3+ was released from both the N‐ and C‐lobes at pH 5.0 in the presence of ATP in ~ 3 minutes as determined by time‐course reaction. Molecular modeling studies identified a putative 25 amino acid ATP binding channel in the N‐lobe of human serum transferrin. These conserved amino acids were identified on each open face of the NI/NII subdomains of apo‐transferrin with the channel forming upon NI/NII subdomain closure as Fe3+ is acquired. At neutral pH ATP bound approximately 9 Å from Fe3+ in the metal center. At acidic pH the protein conformational change positioned ATP immediately adjacent to Fe3+ for rapid chelation. In that ATP is an extracellular, allosteric signaling molecule a mechanism of Fe3+ release from Fe3+‐transferrin is proposed. Here, extracellular ATP is acquired by transferrin in the serum at neutral pH where chelation does not occur. Upon binding to the transferrin receptor and internalization into an acidic endosome, a pH induced conformational change along with positive heterotropic effects occur to allow ATP to efficiently chelate Fe3+. We conclude that transferrin is an allosteric protein, ATP is an allosteric modulator and effective chelator, and that ATP is likely a major Fe3+ carrier in higher organisms.Support or Funding InformationState of Iowa grant GIVFThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.