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Structurally engineered cytochromes c with novel ligand-binding properties

Citation

Bren, Kara Lynne (1996) Structurally engineered cytochromes c with novel ligand-binding properties. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechTHESIS:02042014-091755725

Abstract

Semisynthesis of horse heart cytochrome c and site-directed mutagenesis of Saccharomyces cerevisiae (S. c.) iso-1-cytochrome c have been utilized to substitute Ala for the cytochrome c heme axial ligand Met80 to yield ligand-binding proteins (horse heart Ala80cyt c and S.c. Ala80cyt c) with spectroscopic properties remarkably similar to those of myoglobin. Both species of Fe(II)Ala80cyt c form exceptionally stable dioxygen complexes with autoxidation rates 10-30x smaller and O2 binding constants ~ 3x greater than those of myoglobin. The resistance of O2-Fe(II)Ala80cyt c to autoxidation is attributed in part to protection of the heme site from solvent as exhibited by the exceptionally slow rate of CO binding to the heme as well as the low quantum yield of CO photodissociation.

UV/vis, EPR, and paramagnetic NMR spectroscopy indicate that at pH 7 the Fe(III)Ala80cyt c heme is low-spin with axial His-OH- coordination and that below pH ~6.5, Fe(III)Ala80cyt cis high-spin with His-H2O heme ligation. Significant differences in the pH dependence of the 1H NMR spectra of S.c. Fe(III)Ala80cyt c compared to wild-type demonstrate that the axial ligands influence the conformational energetics of cytochrome c.

1H NMR spectroscopy has been utilized to determine the solution structure of the cyanide derivative of S.c. Fe(III)Ala80cyt c. 82% of the resonances in the 1H NMR spectrum of S.c. CN-Fe(III)Ala80cyt c have been assigned through 1D and 2D experiments. The RMSD values after restrained energy minimization of the family of 17 structures obtained from distance geometry calculations are 0.68 ± 0.11 Å for the backbone and 1.32 ± 0.14 Å for all heavy atoms. The solution structure indicates that a tyrosine in the "distal" pocket of CN-Fe(III)Ala80cyt c forms a hydrogen bond with the Fe(III)-CN unit, suggesting that it may play a role analogous to that of the distal histidine in myoglobin in stabilizing the dioxygen adduct.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:heme, NMR, protein engineering, semisynthesis
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:
  • Gray, Harry B. (chair)
  • Rees, Douglas C.
  • Barton, Jacqueline K.
Defense Date:14 November 1995
Non-Caltech Author Email:kara.bren (AT) rochester.edu
Record Number:CaltechTHESIS:02042014-091755725
Persistent URL:http://resolver.caltech.edu/CaltechTHESIS:02042014-091755725
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:8062
Collection:CaltechTHESIS
Deposited By: Benjamin Perez
Deposited On:04 Feb 2014 17:45
Last Modified:07 Feb 2014 18:00

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