Albumin is the major transport protein in blood for Zn 2؉ , a metal ion required for physiological processes and recruited by various drugs and toxins. However, the Zn 2؉ -binding site(s) on albumin is ill-defined. We have analyzed the 18 x-ray crystal structures of human albumin in the PDB and identified a potential five-coordinate Zn site at the interface of domains I and II consisting of N ligands from His-67 and His-247 and O ligands from Asn-99, Asp-249, and H 2O, which are the same amino acid ligands as those in the zinc enzymes calcineurin, endonucleotidase, and purple acid phosphatase. The site is preformed in unliganded apo-albumin and highly conserved in mammalian albumins. We have used 111 Cd NMR as a probe for Zn 2؉ binding to recombinant human albumin. We show that His-67 3 Ala (His67Ala) mutation strongly perturbs Cd 2؉ binding, whereas the mutations Cys34Ala, or His39Leu and Tyr84Phe (residues which may H-bond to Cys-34) have no effect. Weak Cl ؊ binding to the fifth coordination site of Cd 2؉ was demonstrated. Cd 2؉ binding was dramatically affected by high fatty acid loading of albumin. Analysis of the x-ray structures suggests that fatty acid binding to site 2 triggers a spring-lock mechanism, which disengages the upper (His-67͞Asn-99) and lower (His-247͞Asp-249) halves of the metal site. These findings provide a possible mechanism whereby fatty acids (and perhaps other small molecules) could influence the transport and delivery of zinc in blood.

Most blood plasma zinc is bound to albumin, but the structure of the binding site has not been determined. Zn K-edge extended x-ray absorption fine structure spectroscopy and modeling studies show that the major

Cys34 in domain I of the three‐domain serum protein albumin is the binding site for a wide variety of biologically and clinically important small molecules, provides antioxidant activity, and constitutes the largest portion of free thiol in blood. Analysis of X‐ray structures of albumin reveals that the loop containing Tyr84 occurs in multiple conformations. In structures where the loop is well defined, there appears to be an H‐bond between the OH of Tyr84 and the sulfur of Cys34. We show that the reaction of 5,5′‐dithiobis(2‐nitrobenzoic acid) (DTNB) with Tyr84Phe mutant albumin is approximately four times faster than with the wild‐type protein between pH 6 and pH 8. In contrast, the His39Leu mutant reacts with DTNB more slowly than the wild‐type protein at pH < 8, but at a similar rate at pH 8. Above pH 8 there is a dramatic increase in reactivity for the Tyr84Phe mutant. We also report 1H NMR studies of disulfide interchange reactions with cysteine. The tethering of the two loops containing Tyr84 and Cys34 not only appears to control the redox potential and accessibility of Cys34, but also triggers the transmission of information about the state of Cys34 throughout domain I, and to the domainI/II interface.

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