Kinetic studies on isomerization of ferricytochrome c in alkaline and acid pH ranges by the circular dichroism stopped-flow method

doi:10.1016/0005-2795(80)90120-8

Biochimica et Biophysica Acta (BBA) - Protein Structure

Volume 626, Issue 2, 16 December 1980, Pages 265-276

https://doi.org/10.1016/0005-2795(80)90120-8 Get rights and content

Abstract

The isomerization of horse-heart ferricytochrome c caused by varying pH was kinetically studied by using circular dichroism (CD) and optical absorption stopped-flow techniques.

In the pH range of 7–13, the existence of the three different forms of ferricytochrome c (pH < 10, pH 10–12, and pH > 12) was indicated from the statical difference CD spectra. On the basis of analyses of the stopped-flow traces in the near-ultraviolet and Soret wavelength regions, the isomerization of ferricytochrome c from neutral form to the above three alkaline forms was interpreted as follows: (1) below pH 10, the replacement of the intrinsic ligand of methionine residue by lysine residue occurs; (2) between pH 10 and 12, the uncoupling of the polypeptide chain from close proximity of the heme group occurs first, followed by the interconversion of the intrinsic ligands; and (3) above pH 12, hydroxide form of ferricytochrome c is formed, though the replacement of the intrinsic ligand by extrinsic ligands may occur via different routes from those below pH 12.

The CD changes at 288 nm and in the Soret region caused by the pH-jump (down) from pH 6.0 to 1.6 were compared with the appearance of the 620-nm absorption band ascribed to the formation of the high-spin form of ferricytochrome c. Both CD and absorption changes indicated that the isomerization at pH 1.6 consisted of two processes: one proceeded within the dead-time (about 2 ms) of the stopped-flow apparatus and the other proceeded at a determinable rate with the apparatus. On the basis of these results, the isomerization of ferricytochrome c at pH 1.6 was explained as follows: (1) the transition from the low-spin form to the high-spin form occurs within about 2 ms, the dead-time of the stopped-flow apparatus; and (2) the polypeptide chain is unfolded after the formation of the high-spin form.

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The kinetics of the alkaline transition in mitochondrial Cc have been extensively studied and the mechanistic features can be described by a simple model depicted in Scheme 1, whereby deprotonation of a group termed the trigger (pKH1), initiates a conformational change that leads to the substitution of the Met80 ligand with a Lys residue [54]. At higher pH values (>10) a second rapid phase (kobs2), in addition to the slow phase (kobs1) has been reported for hCc, several of its variants and horse heart Cc [25,55–58]. For the A51V variant this rapid phase is also observed (Fig. 6A).
Mitochondrial cytochrome c is associated with electron transfer in the respiratory chain and in apoptosis. Four cytochrome c variants have been identified in families that suffer from mild autosomal dominant thrombocytopenia, a platelet disorder associated with increased apoptosis. Three out of the four substitutions, G41S, Y48H and A51V are located on the 40–57 Ω-loop. The G41S and Y48H variants perturb key physicochemical and dynamic properties that result in enhanced functional features associated with apoptotic activity. Herein we characterise the ferric A51V variant. We show by chemical denaturation that this variant causes the native state to be destabilized. Through azide binding kinetics, the population of a pentacoordinate heme form, whereby the Met80 axial ligand is dissociated, is estimated to be of equal magnitude to that found in the Y48H variant. This pentacoordinate form gives rise to peroxidase activity, which despite the similar pentacoordinate population of the A51V variant to that of the Y48H variant, the peroxidase activity of the A51V variant is suppressed. Far-UV circular dichroism spectroscopy and pH jump studies, suggest that a combination of structural and dynamic features in addition to the population of the pentacoordinate form regulate peroxidase activity in these disease variants. Additionally, the steady-state ratio of ferric/ferrous cytochrome c when in turnover with cytochrome c oxidase has been investigated for all 40–57 Ω-loop variants. These studies show that the lower pK_a of the alkaline transition for the disease causing variants increases the ferric to ferrous heme ratio, indicating a possible influence on respiration in vivo.
Humanlike substitutions to Ω-loop D of yeast iso-1-cytochrome c only modestly affect dynamics and peroxidase activity
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Citation Excerpt :
It also grew in abruptly above pH 9.5 with an apparent pKa of 10.75 and n ~ 4. High pH fast phases on the 10–50 ms time scale for the alkaline transition have been reported previously for mammalian cytochromes c [49–53]. These phases have been attributed to unfolding of the least stable substructure of Cytc [49], formation of a weakened heme-Met80 bond [51,52] or displacement of Met80 by hydroxide [51].
Structural studies of yeast iso-1-cytochrome c (L.J. McClelland, T.-C. Mou, M.E. Jeakins-Cooley, S.R. Sprang, B.E. Bowler, Proc. Natl. Acad. Sci. U.S.A. 111 (2014) 6648–6653) show that modest movement of Ω-loop D (residues 70–85, average RMSD versus the native structure: 0.81 Å) permits loss of Met80-heme ligation creating an available coordination site to catalyze the peroxidase activity mediated by cytochrome c early in apoptosis. However, Ala81 and Gly83 move significantly (RMSDs of 2.18 and 1.26 Å, respectively). Ala81 and Gly83 evolve to Ile and Val, respectively, in human cytochrome c and peroxidase activity decreases 25-fold relative to the yeast protein at pH 7. To test the hypothesis that these residues evolved to restrict the peroxidase activity of cytochrome c, A81I and G83V variants of yeast iso-1-cytochrome c were prepared. For both variants, the apparent pK_a of the alkaline transition increases by 0.2 to 0.3 relative to the wild type (WT) protein and the rate of opening the heme crevice is slowed. The cooperativity of acid unfolding is decreased for the G83V variant. At pH 7 and 8, the catalytic rate constant, k_cat, for the peroxidase activity of both variants decreases relative to WT, consistent with the effects on alkaline isomerization. Below pH 7, the loss in the cooperativity of acid unfolding causes k_cat for peroxidase activity to increase for the G83V variant relative to WT. Neither variant decreases k_cat to the level of the human protein, indicating that other residues also contribute to the low peroxidase activity of human cytochrome c.
Protein dynamics and function: Making new strides with an old warhorse, the alkaline conformational transition of cytochrome c
2011, Coordination Chemistry Reviews
Citation Excerpt :
When inspecting Scheme 1, the simplest assignment for the triggering group which generates KH is the lysine side chain [91,92], since the amine functionality will only bind to the heme iron in the deprotonated state. Thermodynamic and kinetic experiments have provided a pKH that is near 11 and consistent with lysine deprotonation [31,68,87,90,93]. One study utilizing a set of lysine-to-alanine variants identified a strong correlation between the lowest theoretical pKa calculated for the available lysine heme ligands and the pKapp observed during alkaline conformational transition experiments measured by cyclic voltammetry [91].
Protein dynamics is intimately linked to function. In metalloproteins, dynamics are often coupled to redox activity, ligand binding and enzyme function. We provide a concise overview of the field and then focus on the use of the alkaline conformer of cytochrome c as a model system to probe the factors that control the conformational dynamics of proteins in general and metalloproteins in particular. We consider the effects of ligands on metal-mediated dynamics, the interplay between intrinsic metal-ligand dynamics and barriers imposed by the protein scaffold itself, and the effects of local and overall protein stability on dynamics. Discussed within are the collected results from equilibrium thermodynamic methods, pH jump kinetics and conformationally gated redox reactions between small inorganic reagents and metalloproteins used as a means to probe conformational switching in metalloproteins.
Folding units govern the cytochrome c alkaline transition
2003, Journal of Molecular Biology
The alkaline transition of cytochrome c is a model for protein structural switching in which the normal heme ligand is replaced by another group. Stopped flow data following a jump to high pH detect two slow kinetic phases, suggesting two rate-limiting structure changes. Results described here indicate that these events are controlled by the same structural unfolding reactions that account for the first two steps in the reversible unfolding pathway of cytochrome c. These and other results show that the cooperative folding-unfolding behavior of protein foldons can account for a variety of functional activities in addition to determining folding pathways.
The direct electrochemistry of cryo-hydrogel immobilized myoglobin at a glassy carbon electrode
1995, Journal of Electroanalytical Chemistry
A cryo-hydrogel membrane (CHM) immobilized at a glassy carbon (GC) electrode is reported for the direct electron transfer of redox proteins. The most attractive characteristics of this CHM were its hydrophilic micro-environment for incorporated proteins to retain their activities, its high ability for protection against interference of denatured and adsorbed proteins at the electrode, its potential applications for various proteins or enzymes, as well as its high mechanical strength and thermal stability. A clear well developed and stable redox wave was obtained for commercially available horse heart myoglobin without further purification, giving a peak to peak separation ΔE_p = 93 mV at 5 mV s⁻¹ and the formal electrode potential E⁰′ = −0.158 V (vs. Ag|Agcl). The formal heterogeneous electron transfer rate constant was calculated as k⁰′ = 5.7 × 10⁻⁴ cm s⁻¹ at pH 6.5, showing rapid electron transfer was achieved. The pH controlled conformational equilibria, acid state η natural state η basic I state η basic II state, of myoglobin at the CHM GC electrode in the pH range 0–13.8 were also observed and are discussed in detail.
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1989, Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
Urea denaturation of spinach ferredoxin was investigated under various conditions. The existence of a partially denatured form of the protein was indicated by CD and absorption spectra. The urea concentrations at the midpoints of CD changes at 426 and 222 nm reflecting the conversion of the native to partially denatured form were about 2.3 and 2.1 M, respectively, indicating that a significant alteration in the microenvironment of the iron-sulfur cluster occurred concomitantly with that in the ordered secondary structures. The partially denatured form still had the cluster as judged from the visible absorption changes and reverted to the native form upon dilution of urea. The conversion of the native to partially denatured form consisted of two first-order processes, and the rate constant of the fast process was more largely dependent on urea concentration than that of the slow one. The activation energies for the fast and slow processes at 4 M urea were calculated to be 4.6 and 4.3 kcal/mol, respectively. By the addition of NaCl, the partially denatured form at 4 M urea could practically revert to the native form, whereas that at 8 M urea could not. The binding of 2 mol of cations was found to suppress completely the structural changes at 4 M urea. On the other hand, the conversion of the partially to completely denatured form, where the cluster decomposed, was extremely slow and did not follow simple first-order kinetics. The completely denatured form could not be recovered to the native form, even though the urea was diluted. These results lead to the conclusions that (1) electrostatic interactions rather than nonpolar interactions play an important role for maintaining the protein structure of ferredoxin, and (2) the secondary and tertiary ordered structures persist even in denaturants at relatively high concentration.

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Kinetic studies on isomerization of ferricytochrome c in alkaline and acid pH ranges by the circular dichroism stopped-flow method

Abstract

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Biochem. Biophys. Res. Commun.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Methods Enzymol.

J. Biol. Chem.

J. Am. Chem. Soc.