Flexibility of the metal-binding region in apo-cupredoxins

Abstract

Protein-mediated electron transfer is an essential event in many biochemical processes. Efficient electron transfer requires the reorganization energy of the redox event to be minimized, which is ensured by the presence of rigid donor and acceptor sites. Electron transfer copper sites are present in the ubiquitous cupredoxin fold, able to bind one or two copper ions. The low reorganization energy in these metal centers has been accounted for by assuming that the protein scaffold creates an entatic/rack-induced state, which gives rise to a rigid environment by means of a preformed metal chelating site. However, this notion is incompatible with the need for an exposed metal-binding site and protein-protein interactions enabling metallochaperone-mediated assembly of the copper site. Here we report an NMR study that reveals a high degree of structural heterogeneity in the metal-binding region of the nonmetallated Cu A -binding cupredoxin domain, arising from microsecond to second dynamics that are quenched upon metal binding. We also report similar dynamic features in apo-azurin, a paradigmatic blue copper protein, suggesting a general behavior. These findings reveal that the entatic/rack-induced state, governing the features of the metal center in the copper-loaded protein, does not require a preformed metal-binding site. Instead, metal binding is a major contributor to the rigidity of electron transfer copper centers. These results reconcile the seemingly contradictory requirements of a rigid, occluded center for electron transfer, and an accessible, dynamic site required for in vivo copper uptake.metalloproteins | nuclear magnetic resonance | protein dynamics

Loop recognition and copper-mediated disulfide reduction underpin metal site assembly of Cu A in human cytochrome oxidase

“…2B). This selectivity confirms that these loops are indeed crucial for recognition in the copper transfer event (33). On the other hand, Cu(I)-Sco2 shows impaired metal transfer ability but is able to reduce the cysteines of apo-COX II* S-S at a 2:1 stoichiometry.…”

“…The heteronuclear single-quantum correlation (HSQC) spectra of 15 N apo-COX II* 2SH (where 2SH indicates that the Cys ligands are in the reduced state) show upon copper(I) titration (i) chemical shift perturbations of resonances corresponding to residues close to the metal-binding region, and (ii) the appearance of several cross peaks corresponding to residues in the engineered loops (Fig. S2) that were not observed in the spectrum of the metal-free protein, most likely broadened because of conformational exchange processes occurring in the intermediate regime (33). These differences in the NMR spectra of apo and di-Cu(I) forms of COX II* can be exploited to follow copper uptake from the potential protein donors.…”

“…Removal of this transmembrane stretch has allowed the expression of soluble COX II domains from several bacterial oxidases (30,31). Among them, the soluble fragment from Thermus thermophilus COX II (Tt COX II hereafter) is the only one stable for long periods of time and under different conditions (32,33). Instead, no eukaryotic COX II subunit has been expressed in a stable, soluble form to date.…”

“…1) (34). We have proposed that this region, together with two other neighboring loops, could be involved in the interaction with the metallochaperone (33). Based on this hypothesis, we have designed a chimeric protein by grafting the three loops of the human protein into the stable scaffold of the Thermus protein (Fig.…”

Electron Transfer Proteins as Electronic Conductors: Significance of the Metal and Its Binding Site in the Blue Cu Protein, Azurin

“…This rule, first reported in 1938, was based on analyses of electrical transport measurements of oxide semiconductors,28 where it was observed that the activation energy ( E a ) of the conduction is proportional to the temperature‐independent preexponential factor. This means that if the conduction is thermally activated: $I=I_0\exp (-E_{\mathrm{normala}}/k_{\mathrm{normalB}}T)$, the prefactor $I_0\propto \exp (E_{\mathrm{normala}}/k_{\mathrm{normalB}}{T}_{\mathrm{MN}})$, where T MN is the Meyer–Neldel temperature 29.…”