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Reactions of Heme Proteins to Solutions and Crystals

Citation

Tezcan, Faik Akif (2001) Reactions of Heme Proteins to Solutions and Crystals. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechTHESIS:01312019-135136732

Abstract

To assess the effects of heme solvation and iron ligation on reduction potentials in c-type cytochromes, we have examined the redox and ligand-binding properties of microperoxidase-8 (MP8). Methionine-, histidine- and amine-coordination to MP8 were found to account for 130, -40 and -10-mV shifts in the Fe(III/II)-potential, respectively. Our finding that reduction potentials increase with decreasing heme-surface exposure suggests that the protein matrix can further tune the reduction potential by 500 mV through water exclusion from the heme pocket.

The 410-mV upshift in the cytochrome c (cyt c) potential as the heme cofactor is moved from a highly-solvated environment to the protein interior signals a 10-kcal/mol greater stability of the reduced form. Consequently, there exists a range of denaturant concentrations where Fe(II)-cyt c is folded and Fe(III)-cyt c is unfolded. Electron injection into the oxidized protein in this range triggers the folding reaction. Using NADH as a redox photosensitizer, cyt c folding can be initiated within 100 µs. Our results suggest that the folding of cyt c is rate-limited by ligand-substitution events on the iron center.

Due to an increased barrier to ligand substitution, folding of Co(III)-substituted cyt cis 5 orders of magnitude slower than Fe-cyt c. The slow folding kinetics of Co(III)-cyt c have allowed the convenient study of protein dynamics with a variety of spectroscopic techniques, revealing previously unresolved folding pathways involving Lys- and His-misligated populations of the unfolded molecule and extremely long-lived folding intermediates.

Factors that control electron flow between proteins are not well understood, owing to uncertainties in the relative orientations and structures of the reactants during the short time that tunneling occurs. To circumvent this ambiguity, we have measured the kinetics of electron transfer (ET) between native and Zn-substituted tuna cyt c molecules in crystals of known structure. ET rates (320 s-1 for *Zn-cyt c → Fe(III)-cyt c; 2000 s-1 for Fe(II)-cyt c → Zn-cyt c+) over a Zn-Fe distance of 24.1 Å closely match those for intraprotein ET over similar donor-acceptor separations. Our results indicate that van der Waals interactions and water mediated H-bonds provide effective electronic coupling across a protein-protein interface.

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:Restricted to Caltech community only
Research Advisor(s):
  • Gray, Harry B.
Thesis Committee:
  • Rees, Douglas C. (chair)
  • Barton, Jacqueline K.
  • Bercaw, John E.
  • Gray, Harry B.
  • Winkler, Jay Richmond
Defense Date:11 April 2001
Record Number:CaltechTHESIS:01312019-135136732
Persistent URL:http://resolver.caltech.edu/CaltechTHESIS:01312019-135136732
Related URLs:
URLURL TypeDescription
https://doi.org/10.1021/ja982536eDOIArticle adapted for Chapter 1.
https://doi.org/10.1021/bi981933zDOIArticle adapted for Chapter 2.
https://doi.org/10.1021/ja993447kDOIArticle adapted for Chapter 3.
https://doi.org/10.1073/pnas.081072898DOIArticle adapted for Chapter 4.
ORCID:
AuthorORCID
Tezcan, Faik Akif0000-0002-4733-6500
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:11371
Collection:CaltechTHESIS
Deposited By: Benjamin Perez
Deposited On:31 Jan 2019 22:27
Last Modified:31 Jan 2019 22:56

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