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Physical Characterization of the Rack Effect and Hydrogen Bond Networks in Blue Copper Proteins


Machczynski, Michael Christopher (2001) Physical Characterization of the Rack Effect and Hydrogen Bond Networks in Blue Copper Proteins. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/q0yk-sj10.


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.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Chemistry
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Gray, Harry B.
Thesis Committee:
  • Rees, Douglas C. (chair)
  • Gray, Harry B.
  • Bercaw, John E.
  • Richards, John H. (co-chair)
  • Roberts, Richard W.
Defense Date:17 August 2000
Record Number:CaltechTHESIS:03202014-144611863
Persistent URL:
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:8154
Deposited By: Benjamin Perez
Deposited On:21 Mar 2014 14:13
Last Modified:29 Nov 2022 22:33

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