A summary of previous research is presented that indicates that the purpose of a
blue copper protein's fold and hydrogen bond network, aka, the rack effect, enforce a
copper(II) geometry around the copper(I) ion in the metal site. In several blue copper
proteins, the C-terminal histidine ligand becomes protonated and detaches from the
copper in the reduced forms. Mutants of amicyanin from Paracoccus denitrificans were
made to alter the hydrogen bond network and quantify the rack effect by pKa shifts.
The pKa's of mutant amicyanins have been measured by pH-dependent
electrochemistry. P94F and P94A mutations loosen the Northern loop, allowing the
reduced copper to adopt a relaxed conformation: the ability to relax drives the reduction
potentials up. The measured potentials are 265 (wild type), 380 (P94A), and 415 (P94F)
mV vs. NHE. The measured pKa's are 7.0 (wild type), 6.3 (P94A), and 5.0 (P94F). The
additional hydrogen bond to the thiolate in the mutants is indicated by a red-shift in the
blue copper absorption and an increase in the parallel hyperfine splitting in the EPR
spectrum. This hydrogen bond is invoked as the cause for the increased stability of the C-terminal
imidazole.
Melting curves give a measure of the thermal stability of the protein. A
thermodynamic intermediate with pH-dependent reversibility is revealed. Comparisons
with the electrochemistry and apoamicyanin suggest that the intermediate involves the
region of the protein near the metal site. This region is destabilized in the P94F mutant;
coupled with the evidence that the imidazole is stabilized under the same conditions
confirms an original concept of the rack effect: a high energy configuration is stabilized
at a cost to the rest of the protein.</p
