Low-Temperature, Cooperative Conformational Transition within [Zn-Cytochrome C Peroxidase, Cytochrome c] Complexes: Variation with Cytochrome

Judith M. Nocek, Eric D.A. Stemp, Michael G. Finnegan, Thomas I. Koshy, Michael K. Johnson, E. Margoliash*, A. Grant Mauk, Michael Smith, Brian M Hoffman

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

58 Scopus citations

Abstract

Conformational dynamics within the complex between Zn-substituted cytochrome c peroxidase (ZnCcP) and cytochrome c (Cc) has been studied by examining the quenching of the 3ZnP excited state by the ferriheme of Cc. The temperature and solvent dependence of the triplet-state quenching rate constants (kq) show that complexes of ZnCcP with a large set of Fe3+ cytochromes c undergo a transition between a low-temperature state that does not exhibit triplet quenching and a high-temperature state that does. Within the narrow transition range (220 K < T < 250 K), the decay traces for the [ZnCcP, Fe3+Cc] complexes are nonexponential, and outside of this range they are exponential. This behavior is displayed by complexes with Cc(Drosophila melanogaster), Cc(Candida krusei) and a suite of site-directed mutants of Cc(yeast iso-1) where position 82 contains either an aliphatic (Met, Ser, Leu, or Ile) or an aromatic (Phe) residue. Above 250 K, kq varies strongly among these complexes and decreases sharply with the concentration of cosolvent (EG = ethylene glycol), apparently because of increasing viscosity, while both the breadth of the transition range and its midpoint vary little within this class. MCD and optical spectra between ambient and 4 K rule out the trivial explanation that the transition might reflect changes in the coordination state, and the invariance of kq with a 10-fold increase in [Cc] shows that the proteins remain bound as a complex upon cooling. As the midpoint and breadth of the transition are unaffected by changes in percent EG, the transition does not appear to arise from a solvent-driven process. Instead, we propose that, at ambient temperatures, the binding interface of the [CcP, Cc] complex undergoes rapid dynamic rearrangements between the subset of conformers that exhibit 3ZnP quenching and the subset that does not. Below the transition range, the complex exists in the latter form, and it is suggested that, upon heating, there is a cooperative loosening of the binding interactions within the interface. We present an heuristic description of the complex based on the statistical mechanical description of the cooperative helix-coil transition in poly(amino acids). In contrast, members of a second class of complexes, those with Cc(horse), Cc(tuna), and Cc(rat), have low quenching rate constants (kq ≈ 40 s−1 at ambient) that decrease smoothly to ką = 0 s−1 by 250 K. Furthermore, kq for these complexes shows little dependence upon either solvent or cytochrome, and the triplet decay traces remain exponential at all temperatures.

Original languageEnglish (US)
Pages (from-to)6822-6831
Number of pages10
JournalJournal of the American Chemical Society
Volume113
Issue number18
DOIs
StatePublished - Aug 1 1991

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

Fingerprint

Dive into the research topics of 'Low-Temperature, Cooperative Conformational Transition within [Zn-Cytochrome C Peroxidase, Cytochrome c] Complexes: Variation with Cytochrome'. Together they form a unique fingerprint.

Cite this