Energetic Mechanism of Cytochrome c-Cytochrome c Oxidase Electron Transfer Complex Formation under Turnover Conditions Revealed by Mutational Effects and Docking Simulation

Sato, Wataru; Hitaoka, Seiji; Inoue, Kaoru; Imai, Mizue; Saio, Tomohide; Uchida, Takeshi; Shinzawa‐Itoh, Kyoko; Yoshizawa, Kazunari; Ishimori, Koichiro

doi:10.1074/jbc.m115.708065

Cited by 23 publications

(38 citation statements)

References 60 publications

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“…Amino-acid residues included in the catalytic binding were assigned based on the Cyt.c-CcO complex structure equilibrated in a solution in which the enzyme exerts its normal catalytic activity (Yonetani & Ray, 1965). The interactions between Cyt.c and CcO elucidated by this crystallographic study are consistent with those revealed for the enzyme-substrate complex under turnover conditions by previous experimental studies involving chemical modifications and kinetics (Ferguson-Miller et al, 1978) or solution NMR and kinetics for complexes containing wild-type and mutant Cyt.c proteins (Sakamoto et al, 2011;Sato et al, 2016).…”

Section: Catalytic Binding Sitessupporting

confidence: 89%

“…Recent site‐directed mutagenesis and kinetics studies of Cyt. c indicated that the ET activities of K13L, K86L/K87L, and K7L/K8L mutants are significantly lower than that of the wild‐type protein (Sato et al , ). The side chains of Lys 8 , Gln 12 , Lys 13 , and Lys 87 of Cyt.…”

Section: Resultsmentioning

confidence: 99%

“…On the basis of chemical modification and kinetic studies (Ferguson-Miller et al, 1978), three lysine residues, Lys 8 , Lys 13 , and Lys 87 , were predicted to interact with CcO. Recent site-directed mutagenesis and kinetics studies of Cyt.c indicated that the ET activities of K13L, K86L/K87L, and K7L/K8L mutants are significantly lower than that of the wild-type protein (Sato et al, 2016 A previous NMR study (Sakamoto et al, 2011) detected structural changes in several hydrophobic amino-acid residues of Cyt.c upon the docking of two proteins, and the authors of that study concluded that Cyt.c interacted with CcO via its non-polar surface surrounding the heme cleft, as in the cytochrome bc 1 complex (Cyt.bc 1 )-Cyt.c (Lange & Hunte, 2002) and Cyt.c-cytochrome c peroxidase (CcP) complexes (Jasion et al, 2012). By contrast, our crystal structure of the Cyt.c-CcO complex has no inter-molecular interactions between hydrophobic amino acids with an inter-atomic distance < 5 Å .…”

Section: Catalytic Binding Sitesmentioning

confidence: 99%

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Complex structure of cytochrome c–cytochrome c oxidase reveals a novel protein–protein interaction mode

et al. 2016

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Mitochondrial cytochrome c oxidase (CcO) transfers electrons from cytochrome c (Cyt.c) to O2 to generate H2O, a process coupled to proton pumping. To elucidate the mechanism of electron transfer, we determined the structure of the mammalian Cyt.c–CcO complex at 2.0‐Å resolution and identified an electron transfer pathway from Cyt.c to CcO. The specific interaction between Cyt.c and CcO is stabilized by a few electrostatic interactions between side chains within a small contact surface area. Between the two proteins are three water layers with a long inter‐molecular span, one of which lies between the other two layers without significant direct interaction with either protein. Cyt.c undergoes large structural fluctuations, using the interacting regions with CcO as a fulcrum. These features of the protein–protein interaction at the docking interface represent the first known example of a new class of protein–protein interaction, which we term “soft and specific”. This interaction is likely to contribute to the rapid association/dissociation of the Cyt.c–CcO complex, which facilitates the sequential supply of four electrons for the O2 reduction reaction.

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Section: Catalytic Binding Sitessupporting

confidence: 89%

Section: Resultsmentioning

confidence: 99%

Section: Catalytic Binding Sitesmentioning

confidence: 99%

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Complex structure of cytochrome c–cytochrome c oxidase reveals a novel protein–protein interaction mode

et al. 2016

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“…Native and 15 N-labeled human Cyt c were expressed in Escherichia coli and purified as previously described [11,12]. Briefly, Rosetta2(DE3)pLysS (Novagen, Madison, WI) cells transformed with the plasmids containing human Cyt c [25] DNA were inoculated in 5 mL 2xYT medium and grown overnight.…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

Polyethylene glycol promotes autoxidation of cytochrome c

Sato

Uchida

Saio

et al. 2018

Biochimica et Biophysica Acta (BBA) - General Subjects

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Cytochrome c (Cyt c) was rapidly oxidized by molecular oxygen in the presence, but not absence of PEG. The redox potential of heme c was determined by the potentiometric titration to be +236 ± 3 mV in the absence of PEG, which was negatively shifted to +200 ± 4 mV in the presence of PEG. The underlying the rapid oxidation was explored by examining the structural changes in Cyt c in the presence of PEG using UV-visible absorption, circular dichroism, resonance Raman, and fluorescence spectroscopies. These spectroscopic analyses suggested that heme oxidation was induced by a modest tertiary structural change accompanied by a slight shift in the heme position (<1.0 Å) rather than by partial denaturation, as is observed in the presence of cardiolipin. The near-infrared spectra showed that PEG induced dehydration from Cyt c, which triggered heme displacement. The primary dehydration site was estimated to be around surface-exposed hydrophobic residues near the heme center: Ile81 and Val83. These findings and our previous studies, which showed that hydrated water molecules around Ile81 and Val83 are expelled when Cyt c forms a complex with CcO, proposed that dehydration of these residues is functionally significant to electron transfer from Cyt c to CcO.

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“…To oxidize the residual ferrous form, horse heart Cyt c (Merck Millipore, Darmstadt, Germany) was treated with potassium ferricyanide (Wako Pure Chemical Industries Ltd.) and was purified on an Amicon ultrafiltration using 5-kDa cutoff membranes to remove excess oxidants. The expression vector for the Cyt c mutant containing Gln at the His26 position was constructed as per Sato, W. et al [28]. The protein was dissolved in 50 mM Tris-HCl buffer (pH 7.5) at various activities of urea (molecular biology grade from Kanto Chemical Co., Inc., Tokyo, Japan) or GdnHCl (biochemistry grade from Kanto Chemical Co.) before the experiments.…”

Section: Determination Of Volume Changes In Protein Unfoldingmentioning

confidence: 99%

Uncovering dehydration in cytochrome <i>c</i> refolding from urea- and guanidine hydrochloride-denatured unfolded state by high pressure spectroscopy

2019

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To investigate the dehydration associated with protein folding, the partial molar volume changes for protein unfolding (ΔV u ) in cytochrome c (Cyt c) were determined using high pressure absorption spectroscopy. ΔV u values for the unfolding to urea-and guanidine hydrochloride (GdnHCl)-denatured Cyt c were estimated to be 56±5 and 29±1 mL mol -1 , respectively. Considering that the volume change for hydration of hydrophobic groups is positive and that Cyt c has a covalently bonded heme, a positive ΔV u reflects the primary contribution of the hydration of heme. Because of the marked tendency of guanidium ions to interact with hydrophobic groups, a smaller number of water molecules were hydrated with hydrophobic groups in GdnHCl-denatured Cyt c than in urea-denatured Cyt c, resulting in the smaller positive ΔV u . On the other hand, urea is a relatively weak denaturant and urea-denatured Cyt c is not completely hydrated, which retains the partially folded structures. To unfold such partial structures, we introduced a mutation near the heme binding site, His26, to Gln, resulting in a negatively shifted ΔV u (4±2 mL mol -1 ) in urea-denatured Cyt c. The formation of the more solvated and less structured state in the urea-denatured mutant enhanced hydration to the hydrophilic groups in the unfolding process. Therefore, we confirmed the hydration of amino acid residues in the protein unfolding of Cyt c by estimating ΔV u , which allows us to discuss the hydrated structures in the denatured states of proteins.In aqueous solutions, linear protein polypeptide chains decrease in entropy and collapse into a globule to minimize the surface area that is exposed to the solvent. The folded state is a low-entropy subensemble in all possible collapsed globular conformations for the protein chain. In contrast, the unfolded state is an ensemble that is not or much less structured and has higher entropy than the folded state does. For folding to be beneficial, the folded state must be sufficiently and energetically favorable to overwhelm the higher entropy associated with structural disorder within the globular phase. The major energetic driving force originates from the van der Waals, hydrogen bonding, and electrostatic interactions, both within the polypeptide chain and between the chain andThe high pressure absorption spectroscopy revealed that the partial volume changes for the unfolding of cytochrome c (Cyt c) to the urea-and guanidine hydrochloride (GdnHCl)-denatured unfolded states (ΔV u ) were positive, reflecting hydration to hydrophobic heme; however, a more positively shifted ΔV u was observed for urea-denatured Cyt c. Introduction of the mutation near the axial ligand induced more drastic changes in the hydrated structure of the urea-denatured Cyt c, suggesting that the hydrated structure in the unfolded state depends on the denaturant. Our approach enables us to examine the dehydration associated with protein folding and hydration structures in the unfolded states.

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Energetic Mechanism of Cytochrome c-Cytochrome c Oxidase Electron Transfer Complex Formation under Turnover Conditions Revealed by Mutational Effects and Docking Simulation

Cited by 23 publications

References 60 publications

Complex structure of cytochrome c–cytochrome c oxidase reveals a novel protein–protein interaction mode

Complex structure of cytochrome c–cytochrome c oxidase reveals a novel protein–protein interaction mode

Polyethylene glycol promotes autoxidation of cytochrome c

Uncovering dehydration in cytochrome <i>c</i> refolding from urea- and guanidine hydrochloride-denatured unfolded state by high pressure spectroscopy

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