Urea denaturation of horse heart ferricytochrome c. Equilibrium studies and characterization of intermediate forms

Abstract

Equilibrium studies of the urea denaturation of horse heart ferricytochrome c, pH 7.0, through alteration of the absolute extinction of the 695-nm band and of the fluorescence efficiency of the tryptophan side chain, have been reported. The denaturation profiles using the two probes have been analyzed in terms of similarities and differences as well as to determine the nature of the intermediate forms. The intermediate forms in the 4-4.5 M urea concentration range have been further characterized by circular dichroism spectroscopy in the Soret and the intrinsic absorption regions, stability to temperature and pH, and reactivity of the methionine and histidine side chains to bromoacetic acid. The scheme N + XI + X2 D, in which N and D are the 0 and 9 M urea forms and XI and X2 are the two intermediate forms,with midconcentrations of urea of 2-2.5, 6.2, and 6.9 M, respectively, for the three transitions, is proposed as an explanation of the observed denaturation profiles of the protein.The N to XI transition is seen in the enhanced absorptivity of the 695-nm band and the further quenching of tryptophan fluorescence. Form X, exhibits lowered temperature and pH stability, a nativelike intrinsic CD spectrum, and reactivity of both His-18 and Met-80 to bromoacetic acid. The X I to X2transition is apparent only in the 695-nm absorptivity-urea profile, while the X2 to D transition is discerned from both the enhancement of tryptophan fluorescence and the final quenching of the 695-nm absorptivity. The extent of alteration T e structural, conformational, and possibly functional aspects of enzymes can be better understood from a detailing of the folding and unfolding processes. In the case of the constituents of the electron-transport chain, cytochrome c is the only component susceptible to such investigation, primarily because of its availability in a pure, well-defined form and the wealth of information regarding its structure,

A model for the misfolded bis-His intermediate of cytochrome c: the 1–56 N-fragment

“…The fragment shows the same spectroscopic characteristics as ferric and ferrous cyt c under partially denaturing conditions when a non native histidine (either His26 or His33) ligand is known to replace Met80 [9]. Moreover, the misligated state of cyt c and the N-fragment have very similar dichroic spectra both in the Soret and in the far-UV regions [16,[38][39][40]. Nevertheless, the peptide chain shields the heme group from solvent and the titration (as a function of pH) of the ferric fragment shows a similar behavior to the GuHCl-denatured protein albeit with lower pK a .…”

Redox thermodynamics of cytochromes c subjected to urea induced unfolding

“…2a, 3a; Tables 2, 3 [15,[22][23][24] for cytochrome c at room temperature (see above) whereas X HT is a different form which should not differ much from the low-T species, on the basis of the similar electrochemical behavior observed.…”

“…However, here we adopt the model by Myer et al [15] to interpret the electrochemical behavior of cytochrome c in the presence of urea without specific structural considerations which would be rather speculative. Therefore, the E°0 shift at low urea concentration can in principle be correlated with the progressive unfolding of the N-yellow X loop [21], but no further info on the role of this foldon can be gained.…”

“…Significant insight into the functional properties of cytochrome c can also be gained from the investigation of the folding and unfolding process, in particular the mechanisms controlling the formation of the functionally active three-dimensional structure of the protein [9][10][11][12][13][14]. Myer et al [15] proposed a denaturation mechanism summarized by the scheme:…”

“…The residues Lys72, Lys73 and Lys79 are distributed around the heme crevice and are involved in molecular recognition with the redox partners and pH-induced conformational transitions [1-3, 26, 27]. The replacement of these three positively charged residues with neutral ones perturbates the charge distribution on the crevice, affecting the network of hydrogen bonds that is responsible for the stabilization of the protein 3D structure [15,24].…”