The copper(II) binding centres of carbonic anhydrase are differently affected by reductants that ensure the redox intracellular environment

Abstract

Copper is involved in several biological processes. The static and labile copper pools are controlled by means of a network of influx and efflux transporters, storage proteins, chaperones, transcription factors and small molecules as glutathione (GSH), which contributes to the cell reducing environment. To follow the fate of intracellular copper labile pool, a variant of human apocarbonic anhydrase has been proposed as fluorescent probe to monitor cytoplasmic Cu²⁺. Aware that in this cellular compartment copper ion is present as Cu⁺, electron spin resonance technique (ESR) was used to ascertain whether (bovine or human) carbonic anhydrase (CA) was able to accommodate Cu⁺ in the same sites occupied by Cu²⁺, in the presence of naturally occurring reducing agents such as ascorbate and GSH. Our ESR results on Cu²⁺ complexes with CA allow for a complete characterization of the two metal binding sites of the protein in solution. The use of the reported affinity constants of zinc in the catalytic site and of Cu²⁺ in the peripheral and catalytic site, allow us to obtain the speciation of copper species mimicking the spectroscopic study conditions. The different Cu²⁺ coordination features in the catalytic and the peripheral (the N-terminus cleft mouth) binding sites influence the chemical reduction effect of the two main naturally occurring reductants. Ascorbate reversibly reduces the Cu²⁺ complex with CA, while glutathione irreversibly induces the formation of Cu²⁺ complex with its oxidized form (GSSG). Our results questioned the use of CA as intracellular Cu²⁺ sensor. Furthermore, translating these findings to intracellular environment, the conversion of GSH in GSSG can significantly alter the metallostasis.