RESUMO
Copper is a ubiquitous metal in biology that, among other functions, is implicated in enzymatic redox catalysis and in protein electron transfer (ET). When it comes to ET, copper sites are found in two main forms, mononuclear type 1 (T1) and binuclear CuA sites, which share a common cupredoxin fold. Other relevant copper sites are the so-called type 2 (T2), which are more resilient to undergo direct electrochemistry and are usually involved in catalysis. Here we report the electrochemical and spectroscopic characterization of a novel T2-like copper site engineered following the loop swapping strategy. The ligand loop sequence of the newly discovered T1 copper site from Nitrosopumilus maritimus was introduced into the CuA scaffold from Thermus thermophilus yielding a chimeric protein that shows spectroscopic features different from both parental proteins, and resemble those of red T2 copper sites, albeit with a shorter Cu-S(Cys) bond length. The novel T2 site undergoes efficient direct electrochemistry, which allows performing temperature-dependent cyclic voltammetry studies. The obtained results reveal that this chimera constitutes the first example of a copper protein with entropically controlled reduction potential, thereby contrasting the enthalpic supremacy observed for all other copper sites reported so far. The underlying bases for this entropic control are critically discussed.
Assuntos
Cobre , Thermus thermophilus , Cobre/química , Transporte de Elétrons , Ligantes , Oxirredução , Thermus thermophilus/química , Thermus thermophilus/metabolismoRESUMO
Here we report the spectroscopic and electrochemical characterization of three novel chimeric CuA proteins in which either one or the three loops surrounding the metal ions in the Thermus thermophilus protein have been replaced by homologous human and plant sequences while preserving the set of coordinating amino acids. These conservative modifications mimic basic differences between CuA sites from different organisms and allow for fine tuning the energy gap between alternative electronic ground states of CuA.. This results in a systematic modulation of thermodynamic and kinetic electron transfer (ET) parameters and in the selection of one of two possible redox-active molecular orbitals, which differ in the ET reorganization energy by a factor of 2. Moreover, the ET mechanism is found to be frictionally controlled, and the modifications introduced into the different chimeras do not affect the frictional activation parameter.
Assuntos
Cobre/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Thermus thermophilus/metabolismo , Cobre/química , Cristalografia por Raios X , Técnicas Eletroquímicas , Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons/química , Cinética , Modelos Moleculares , Termodinâmica , Thermus thermophilus/químicaRESUMO
CuA is a binuclear copper site acting as electron entry port in terminal heme-copper oxidases. In the oxidized form, CuA is a mixed valence pair whose electronic structure can be described using a potential energy surface with two minima, σu* and πu, that are variably populated at room temperature. We report that mutations in the first and second coordination spheres of the binuclear metallocofactor can be combined in an additive manner to tune the energy gap and, thus, the relative populations of the two lowest-lying states. A series of designed mutants span σu*/πu energy gaps ranging from 900 to 13 cm-1. The smallest gap corresponds to a variant with an effectively degenerate ground state. All engineered sites preserve the mixed-valence character of this metal center and the electron transfer functionality. An increase of the Cu-Cu distance less than 0.06 Å modifies the σu*/πu energy gap by almost 2 orders of magnitude, with longer distances eliciting a larger population of the πu state. This scenario offers a stark contrast to synthetic systems, as model compounds require a lengthening of 0.5 Å in the Cu-Cu distance to stabilize the πu state. These findings show that the tight control of the protein environment allows drastic perturbations in the electronic structure of CuA sites with minor geometric changes.
Assuntos
Proteínas de Bactérias/química , Complexos de Coordenação/química , Cobre/química , Grupo dos Citocromos b/química , Complexo IV da Cadeia de Transporte de Elétrons/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Grupo dos Citocromos b/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Elétrons , Estrutura Molecular , Engenharia de Proteínas , Subunidades Proteicas/química , Alinhamento de Sequência , Termodinâmica , Thermus thermophilus/enzimologiaRESUMO
Manipulation of the partition function (Q) of the redox center CuA from cytochrome c oxidase is attained by tuning the accessibility of a low lying alternative electronic ground state and by perturbation of the electrostatic potential through point mutations, loop engineering and pH variation. We report clear correlations of the entropic and enthalpic contributions to redox potentials with Q and with the identity and hydrophobicity of the weak axial ligand, respectively.