Basic requirements for a metal-binding site in a protein: The influence of loop shortening on the cupredoxin azurin

doi:10.1073/pnas.0600774103

Basic requirements for a metal-binding site in a protein: The influence of loop shortening on the cupredoxin azurin

^†Institute for Cell and Molecular Biosciences, Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, United Kingdom;
^‡Elitenetzwerk Bayern Macromolecular Science, Mikrobiologie/Proteinkristallographie Laboratories, Universität Bayreuth, D-95440 Bayreuth, Germany; and
^§Max-Planck-Institut für Biochemie, Abteilung Strukturforschung, Am Klopferspitz 18, D-82152 Martinsried, Germany

Edited by Harry B. Gray, California Institute of Technology, Pasadena, CA, and approved March 15, 2006 (received for review January 30, 2006)

Abstract

The main active-site loop of the copper-binding protein azurin (a cupredoxin) has been shortened from C¹¹²TFPGH¹¹⁷SALM¹²¹ to C¹¹²TPH¹¹⁵PFM¹¹⁸ (the native loop from the cupredoxin amicyanin) and also to C¹¹²TPH¹¹⁵PM¹¹⁷. The Cu(II) site structure is almost unaffected by shortening, as is that of the Cu(I) center at alkaline pH in the variant with the C¹¹²TPH¹¹⁵PM¹¹⁷ loop sequence. Subtle spectroscopic differences due to alterations in the spin density distribution at the Cu(II) site can be attributed mainly to changes in the hydrogen-bonding pattern. Electron transfer is almost unaffected by the introduction of the C¹¹²TPH¹¹⁵PFM¹¹⁸ loop, but removal of the Phe residue has a sizable effect on reactivity, probably because of diminished homodimer formation. At mildly acidic pH values, the His-115 ligand protonates and dissociates from the cuprous ion, an effect that has a dramatic influence on the reactivity of cupredoxins. These studies demonstrate that the amicyanin loop adopts a conformation identical to that found in the native protein when introduced into azurin, that a shorter than naturally occurring C-terminal active-site loop can support a functional T1 copper site, that CTPHPM is the minimal loop length required for binding this ubiquitous electron transfer center, and that the length and sequence of a metal-binding loop regulates a range of structural and functional features of the active site of a metalloprotein.

Footnotes

^¶To whom correspondence should be addressed. E-mail: christopher.dennison{at}ncl.ac.uk
Author contributions: C.D. designed research; C.L., S.Y., B.M.M., A.M., M.J.B., and C.D. performed research; S.Y., M.J.B., and C.D. analyzed data; and C.D. wrote the paper.
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 2FTA, 2FT6, 2FT7, and 2FT8).
Abbreviations:

Abbreviations:

AMI,

amicyanin;

AZ,

azurin;

Em,

reduction potential;

ESE,

electron self-exchange;

ET,

electron transfer;

rmsd,

rms deviation;

T1,

type 1;

vis,

visible.