Kinetic Mechanism of Cytochrome c Folding: Involvement of the Heme and Its Ligands

Abstract

The covalently attached heme and its axial ligands not only are essential for the structure and function of cytochrome c but they also play an important role in the folding process. Under typical denaturing conditions (concentrated guanidine hydrochloride or urea near pH 7), one of the axial ligands, His 18, remains bound to the oxidized heme iron, but the second ligand, Met 80, is replaced by a non-native histidine ligand (His 26 or His 33 in horse cytochrome c). Using quenched-flow and NMR methods, hydrogen exchange rates were measured for several individual amide protons in guanidine-denatured horse cytochrome c. The observation of a single highly protected (140-fold) backbone amide, that of His 18, suggests the presence of a persistent H-bond consistent with heme ligation of the His 18 side chain in the unfolded state. Heme absorbance changes induced by rapid acidification of oxidized cytochrome c in 4.5 M guanidine hydrochloride from pH 7.8 to 4.6 or below exhibit two kinetic phases with rates of 110 and 25 s-1, attributed to the dissociation of non-native histidine ligands from the heme in the unfolded state. The kinetics of folding from guanidine-denatured cytochrome c under a variety of initial and final conditions was investigated by stopped-flow methods, using tryptophan fluorescence as a conformational probe and Soret absorbance as a probe for the ligation state of the heme. A fast kinetic phase (80 s-1) accompanied by a major decrease in fluorescence and a minor absorbance change coincides with the formation of a partially folded intermediate with interacting chain termini detected in earlier pulsed NH exchange measurements [Roder, H., Elöve, G. A., & Englander, S. W. (1988) Nature 335, 700]. At neutral pH, an intermediate kinetic phase (1.8 s-1) accounts for 78% of the absorbance change and 47% of the fluorescence change. In contrast, the folding kinetics at pH 5 is dominated by the fast phase, and the amplitude of the intermediate phase is reduced to approximately 10%. The pH-dependent amplitude changes show titration behavior with an apparent pK of approximately 5.7, consistent with the protonation of a single histidine residue. The intermediate phase can also be suppressed by the addition of 20 mM imidazole. Since both of these conditions interfere with histidine ligation, the intermediate kinetic phase is attributed to the presence of a non-native histidine ligand (His 26 or His 33) that can become trapped in a partially folded intermediate.(ABSTRACT TRUNCATED AT 400 WORDS)

Fast folding of cytochromec

“…These results have been interpreted in support of Equation 3, in which heme ligation generates misfolds of horse cytochrome c (Sosnick et al, 1996). Other results tend to support Equation 2, in which heme ligation traps on-pathway folding intermediates kinetically (Elove et al, 1994). When both initial unfolding conditions and final refolding conditions favor strong His-heme ligation, the rate of conversion of kinetically trapped intermediates to native species decreases with increasing Gdn.HC1 (Ikai et al, 1973).…”

“…However, at low pH, where histidine coordination is disfavored, the fraction of folding that occurs on the fastest timescale (10-20 ms) is greatly increased. This shift in amplitude to the fastest folding phase suggests that elimination of non-native histidine-heme interactions enables the majority of the molecules to fold on the fastest (10-20 ms) timescale (Elove et al, 1994;Sosnick et al, 1994). Additional evidence for the involvement of non-native His-heme ligation was provided by the addition of extrinsic ligands to the refolding reaction (Brems & Stellwagen, 1983).…”

Probing the cytochrome c′ folding landscape

“…The shift to pH 5 was driven by the desire to eliminate effects owing to misligation (a major folding trap for cytochrome c) [21]. Although His misligation is not a factor for cyt c′ (the native axial ligand His117 is the only His), the N-terminal amino group as well as sidechains of some of the Lys residues can still coordinate to the heme in the unfolded protein at pH 7 [22,23].…”

“…We conclude that cis-configured prolines are responsible for some of the topological and energetic frustration of the collapsed polypeptide ensemble [2]. Structure of R. palustris cyt c′ (1A7V) [7] showing the heme ligand (His117) and four prolines (21,52, 56, and 69). Absorption changes during the pH titration of pseudo-wild-type (Gln1Ala) R. palustris cyt c′ in 6 M GuHCl.…”

Protein folding and misfolding: mechanism and principles

“…In agreement, intermediates that accumulate in 3-state folding are often seen to harbor misfolding errors, including Cyt c (Elöve et al 1994;Sosnick et al 1994), apoMb (Nishimura et al 2006), Im7 (Capaldi et al 2002), and α-Trp synthase (Wu & Matthews, 2003). This observation provides a nececessary but not a sufficient proof for the blocking role of misfolding because non-native energy-minimizing interactions are likely to be present in any incompletely native state.…”