Perturbation of Pseudomonas cytochrome oxidase by guanidine hydrochloride to detec differential stabilization of the heme d1 and heme c moieties

Paul Horowitz, Barry B. Muhobereac, Karen Falksen, David C. Wharton

Research output: Contribution to journalArticlepeer-review

Abstract

The optical properties of Pseudomonas cytochrome oxidase (ferrocytochrome-c:oxygen oxidoreductase, EC 1.9.3.2) were monitored as a function in guanidine hydrochloride (Gdn · HCl) concentration to probe for differential stabilization of its prosthetic groups, heme d1 and heme c. The protein fluorescence intensity increased with the Gdn · HCl concentration, revealing two transitions, a sharp one between 1.3 and 1.5 M Gdn · HCl, and a second less well defined extending from 2.5 to 4.5 M. Only the transition at the lower Gdn · HCl concentrations was present in titrations followed using the emission maxima. The spectral maximum for native Pseudomonas cytochrome oxidase was at approx. 335 nm and shifted to approx. 350 nm above 2 M Gdn · HCl. The heme d1 adsorbance at 638 nm decreased with increasing [Gdn · HCl], giving a transition at 1.3-1.5 M, and no transition up to 4 M Gdn · HCl when the heme c was monitored at 525 nm. Along with the decrease at 638 nm, an absorption band appeared at 681 nm, suggesting heme d1 release into solution. Fluorescence titration of heme d1-depleted enzyme, prepared by gel filtration, showed a single transition similar to the transition occurring in the intact enzyme at high Gdn· HCl concentrations. Circular dichroism spectra revealed clearly distinguishable transitions for the heme d1 and heme c near 1.5 and 3.0 M Gdn · HCl, respectively. There results suggest that the two hemes are in regions of the protein with different stabilities which may represent distinct structural domains.

Original languageEnglish (US)
Pages (from-to)142-148
Number of pages7
JournalBiochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
Volume871
Issue number2
DOIs
StatePublished - Jun 5 1986
Externally publishedYes

Keywords

  • (Pseudomonas)
  • Cytochrome oxidase stabilityl
  • Protein fluorescence intensity circular dichroism
  • Tertiary structure

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology

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