Redox-controlled backbone dynamics of human cytochrome c revealed by N NMR relaxation measurements

Redox-controlled backbone dynamics in cytochrome c (Cyt c) were revealed by 2D ¹⁵N NMR relaxation experiments. ¹⁵N T₁ and T₂ values and ¹H-¹⁵N NOEs of uniformly ¹⁵N-labeled reduced and oxidized Cyt c were measured, and the generalized order parameters (S²), the effective correlation time for internal motion (τ_e), the ¹⁵N exchange broadening contributions (R_ex) for each residue, and the overall correlation time (τ_m) were estimated by model-free dynamics formalism. These dynamic parameters clearly showed that the backbone dynamics of Cyt c are highly restricted due to the covalently bound heme that functions as the stable hydrophobic core. Upon oxidation of the heme iron in Cyt c, the average S² value was increased from 0.88 ± 0.01 to 0.92 ± 0.01, demonstrating that the mobility of the backbone is further restricted in the oxidized form. Such increases in the S² values were more prominent in the loop regions, including amino acid residues near the thioether bonds to the heme moiety and positively charged region around Lys87. Both of the regions are supposed to form the interaction site for cytochrome c oxidase (CcO) and the electron pathway from Cyt c to CcO. The redox-dependent mobility of the backbone in the interaction site for the electron transfer to CcO suggests an electron transfer mechanism regulated by the backbone dynamics in the Cyt c-CcO system.     

The oxidative nuclease activity of human cytochrome c with mutations in Ω-loop C/D

Natural and artificial nucleases have extensive applications in biotechnology and biomedicine. The exploration of protein with potential DNA cleavage activity also inspires the design of artificial nuclease and helps to understand the physiological process of DNA damage. In this study, we engineered four human cytochrome c (Cyt c) mutants (N52S, N52A, I81N, and I81D Cyt c), which showed enhanced DNA cleavage activity and degradation in comparison with WT Cyt c, especially under acidic conditions. The mechanism assays revealed that the superoxide (O₂^??) plays an important role in the nuclease reaction. The kinetic assays showed that the peroxidase activity of the I81D Cyt c mutant enhanced up to 9-fold at pH 5. This study suggests that the mutations of Ile81 and Asn52 in Ω-loop C/D are critical for the nuclease activity of Cyt c, which may have physiological significance in DNA damage and potential applications in biomedicine.     

Structure analysis and characterization of the cytochrome c-554 from thermophilic green sulfur photosynthetic bacterium Chlorobaculum tepidum

The cytochrome (Cyt) c-554 in thermophilic green photosynthetic bacterium Chlorobaculum tepidum serves as an intermediate electron carrier, transferring electrons to the membrane-bound Cyt c _z from various enzymes involved in the oxidations of sulfide, thiosulfate, and sulfite compounds. Spectroscopically, this protein exhibits an asymmetric α-absorption band for the reduced form and particularly large paramagnetic ¹H NMR shifts for the heme methyl groups with an unusual shift pattern in the oxidized form. The crystal structure of the Cyt c-554 has been determined at high resolution. The overall fold consists of four α-helices and is characterized by a remarkably long and flexible loop between the α3 and α4 helices. The axial ligand methionine has S-chirality at the sulfur atom with its C^εH₃ group pointing toward the heme pyrrole ring I. This configuration corresponds to an orientation of the lone-pair orbital of the sulfur atom directed at the pyrrole ring II and explains the lowest-field ¹H NMR shift arising from the 18¹ heme methyl protons. Differing from most other class I Cyts c, no hydrogen bond was formed between the methionine sulfur atom and polypeptide chain. Lack of this hydrogen bond may account for the observed large paramagnetic ¹H NMR shifts of the heme methyl protons. The surface-exposed heme pyrrole ring II edge is in a relatively hydrophobic environment surrounded by several electronically neutral residues. This portion is considered as an electron transfer gateway. The structure of the Cyt c-554 is compared with those of other Cyts c, and possible interactions of this protein with its electron transport partners are discussed.     

Probing the conformational changes and peroxidase-like activity of cytochrome c upon interaction with iron nanoparticles

Herein, the interaction of iron nanoparticle (Fe-NP) with cytochrome c (Cyt c) was investigated, and a range of techniques such as dynamic light scattering (DLS), zeta potential measurements, static and synchronous fluorescence spectroscopy, near and far circular dichroism (CD) spectroscopy, and ultraviolet–visible (UV–vis) spectroscopy were used to analyze the interaction between Cyt c and Fe-NP. DLS and zeta potential measurements showed that the values of hydrodynamic radius and charge distribution of Fe-NP are 83.95 ± 3.7 nm and 4.5 ± .8 mV, respectively. The fluorescence spectroscopy results demonstrated that the binding of Fe-NP with Cyt c is mediated by hydrogen bonds and van der Waals interactions. Also Fe-NP induced conformational changes in Cyt c and reduced the melting temperature value of Cyt c from 79.18 to 71.33°C. CD experiments of interaction between Fe-NP and Cyt c revealed that the secondary structure of Cyt c with the dominant α-helix structures remained unchanged whereas the tertiary structure and heme position of Cyt c are subjected to remarkable changes. Absorption spectroscopy at 695 nm revealed that Fe-NP considerably disrupt the Fe…S(Met80) bond. In addition, the UV–vis experiment showed the peroxidase-like activity of Cyt c upon interaction with Fe-NP. Hence, the data indicate the Fe-NP results in unfolding of Cyt c and subsequent peroxidase-like activity of denatured species. It was concluded that a comprehensive study of the interaction of Fe-NP with biological system is a crucial step for their potential application as intracellular delivery carriers and medicinal agents.     

YhjA - An Escherichia coli trihemic enzyme with quinol peroxidase activity

The trihemic bacterial cytochrome c peroxidase from Escherichia coli, YhjA, is a membrane-anchored protein with a C-terminal domain homologous to the classical bacterial peroxidases and an additional N-terminal (NT) heme binding domain. Recombinant YhjA is a 50?kDa monomer in solution with three c-type hemes covalently bound. Here is reported the first biochemical and spectroscopic characterization of YhjA and of the NT domain demonstrating that NT heme is His63/Met125 coordinated. The reduction potentials of P (active site), NT and E hemes were established to be ?170?mV, +133?mV and +210?mV, respectively, at pH?7.5. YhjA has quinol peroxidase activity in vitro with optimum activity at pH?7.0 and millimolar range K_M values using hydroquinone and menadiol (a menaquinol analogue) as electron donors (K_M?=?0.6?±?0.2 and 1.8?±?0.5?mM H₂O₂, respectively), with similar turnover numbers (k_cat?=?19?±?2 and 13?±?2?s^?1, respectively). YhjA does not require reductive activation for maximum activity, in opposition to classical bacterial peroxidases, as P heme is always high-spin 6-coordinated with a water-derived molecule as distal axial ligand but shares the need for the presence of calcium ions in the kinetic assays. Formation of a ferryl Fe(IV)?=?O species was observed upon incubation of fully oxidized YhjA with H₂O₂. The data reported improve our understanding of the biochemical properties and catalytic mechanism of YhjA, a three-heme peroxidase that uses the quinol pool to defend the cells against hydrogen peroxide during transient exposure to oxygenated environments.     

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

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.     

Extinction coefficients of cytochromes b559 and c550 of Thermosynechococcus elongatus and Cyt b559/PS II stoichiometry of higher plants

“Reduced minus oxidized” difference extinction coefficients Δ? in the α-bands of Cyt b559 and Cyt c550 were determined by using functionally and structurally well-characterized PS II core complexes from the thermophilic cyanobacterium Thermosynechococcus elongatus. Values of 25.1 ± 1.0 mM⁻¹ cm⁻¹ and 27.0 ± 1.0 mM⁻¹ cm⁻¹ were obtained for Cyt b559 and Cyt c550, respectively. Anaerobic redox titrations covering the wide range from −250 up to +450 mV revealed that the heme groups of both Cyt b559 and Cyt c550 exhibit homogenous redox properties in the sample preparation used, with E_m values at pH 6.5 of 244 ± 11 mV and −94 ± 21 mV, respectively. No HP form of Cyt b559 could be detected. Experiments performed on PS II membrane fragments of higher plants where the content of the high potential form of Cyt b559 was varied by special treatments (pH, heat) have shown that the α-band extinction of Cyt b559 does not depend on the redox form of the heme group. Based on the results of this study the Cyt b559/PSII stoichiometry is inferred to be 1:1 not only in thermophilic cyanobacteria as known from the crystal structure but also in PSII of plants. Possible interrelationships between the structure of the Q_B site and the microenvironment of the heme group of Cyt b559 are discussed.     

HER2 Ile655Val and PTEN IVS4 polymorphisms in patients with breast cancer

Although HER2/PTEN pathway is commonly disrupted in cancer, association of HER2 and PTEN polymorphisms with breast cancer (BC) remains controversial. We investigated the HER2 Ile655 Val and PTEN IVS4 polymorphisms in patients with BC in Turkish population. HER2 Ile655Val (rs 1136201) and PTEN IVS4 (rs 3830675) polymorphisms were determined using polymerase chain reaction-based restriction fragment length polymorphism (PCR–RFLP) in blood samples of 118 BC patients and 118 age-matched healthy controls. We found that the frequency of the Ile/Val genotype of HER2 Ile655Val gene was significantly higher in BC patients (p < 0.009; OR: 1,983 95 % CI: 1.181—3.328). The presence of ATCTT insertion (+/+) genotype at downstream of exon 4 in intron 4 of PTEN IVS4 gene was also associated with 1.83 fold decreased risk of BC development (p < 0.033; OR: 1.83, 95 % CI: 1.11—3.03). Analysis on clinico-pathological parameters showed neither HER2 Ile655Val nor PTEN IVS4 genotypes were not associated with any of the variables (p > 0.05).In conclusion, our findings suggest that the Ile/Val genotype of HER2 and ATCTT insertion (+/+) genotype of PTEN IVS4 gene may play an important role as genetic markers for breast cancer risk, but both genes genotypes may not be useful for predicting tumor prognosis in Turkish population.     

Peroxidase activity of cytochrome c in its compact state depends on dynamics of the heme region

Cytochrome c (cyt c) is a small globular hemoprotein with the main function as an electron carrier in mitochondrial respiratory chain. Cyt c possesses also peroxidase-like activity in the native state despite its six-coordinated heme iron. In this work, we studied the effect of increasing urea concentration in the range from 0 M to 6 M at pH 7 (pH value of the bulk solvent) and pH 5 (pH value close to negatively charged membrane) on peroxidase-like activity of cyt c. We show that peroxidase-like activity, measured by guaiacol oxidation and the ferrous oxidation in xylenol orange methods, correlates with the accessibility of the heme iron, which was assessed from the association rate constant of cyanide binding to cyt c. Cyt c peroxidase-like activity linearly increases in the pre-denaturational urea concentrations (0–4 M) at both studied pHs without an apparent formation of penta-coordinated state of the heme iron. Our results suggest that dynamic equilibrium among the denaturant-induced non-native coordination states of cyt c, very likely due to reversible unfolding of the least stable foldons, is pre-requisite for enhanced peroxidase-like activity of cyt c in its compact state. Dynamic replacement of the native sixth coordination bond of methionine-80 by lysines (72, 73, and 79) and partially also by histidines (26 and 33) provides an efficient way how to increase peroxidase-like activity of cyt c without significant conformational change at physiological conditions.     

Structural and functional studies on the tetraheme cytochrome subunit and its electron donor proteins: the possible docking mechanisms during the electron transfer reaction

The photosynthetic reaction centers (RCs) classified as the group II possess a peripheral cytochrome (Cyt) subunit, which serves as the electron mediator to the special-pair. In the cycle of the photosynthetic electron transfer reactions, the Cyt subunit accepts electrons from soluble electron carrier proteins, and re-reduces the photo-oxidized special-pair of the bacteriochlorophyll. Physiologically, high-potential cytochromes such as the cytochrome c₂ and the high-potential iron–sulfur protein (HiPIP) function as the electron donors to the Cyt subunit. Most of the Cyt subunits possess four heme c groups, and it was unclear which heme group first accepts the electron from the electron donor. The most distal heme to the special-pair, the heme-1, has a lower redox potential than the electron donors, which makes it difficult to understand the electron transfer mechanism mediated by the Cyt subunit. Extensive mutagenesis combined with kinetic studies has made a great contribution to our understanding of the molecular interaction mechanisms, and has demonstrated the importance of the region close to the heme-1 in the electron transfer. Moreover, crystallographic studies have elucidated two high-resolution three-dimensional structures for the RCs containing the Cyt subunit, the Blastochloris viridis and Thermochromatium tepidum RCs, as well as the structures of their electron donors. An examination of the structural data also suggested that the binding sites for both the cytochrome c₂ and the HiPIP are located adjacent to the solvent-accessible edge of the heme-1. In addition, it is also indicated by the structural and biochemical data that the cytochrome c₂ and the HiPIP dock with the Cyt subunit by different mechanisms although the two electron donors utilize the same region for the interactions; cytochrome c₂ is recognized through electrostatic interactions while hydrophobic interactions are important in the HiPIP docking.     

Appearance of Nitrite Reducing Activity of Cytochrome c upon Heat Denaturation

The appearance of NO₂ ^? reducing activity of cytochrome c (Cyt c) upon heat denaturation was investigated with equine heart Cyt c. Denatured equine heart Cyt c (dCyt c), which was treated at 100°C for 30 min, had NO₂ ^? reducing activity in the presence of dithionite and methylviologen in an aqueous solution under anaerobic conditions. In contrast, hemoglobin and myoglobin had no such activity under the same conditions. Using spectroscopic methods, we found that the appearance of this activity in the Cyt c was due to the following intramolecular changes: unfolding of the peptide chain, exposure of the heme, dissociation of the sixth ligand methionine sulfur, and appearance of autoxidizability. The dCyt c catalyzed NO₂ ^? reduction to NH₄ ⁺ via ferrous-NO complexes, and this reaction was a 6-electron and 8-proton reduction. Sepharose-immobilized dCyt c had activity similar strength to that in solution. The resin retained the activity after five uses and even after storage for 1 year. On the basis of these results, we concluded that Cyt c acquired a new catalytic activity upon heat treatment, unlike to other familiar biological molecules.     

The membrane-bound high-molecular-mass cytochromes c from Desulfovibrio gigas and Desulfovibrio vulgaris Hildenborough; EPR and Mössbauer studies

The high-molecular-mass cytochromes c (Hmcs) from the sulfate-reducing bacteria Desulfovibrio gigas and Desulfovibrio vulgaris (Hildenborough) were found to be strongly bound to the cytoplasmic membrane. After detergent solubilization they were shown to be water soluble and to be similar to those previously isolated from the soluble fractions in terms of N-terminal sequence, molecular mass, UV-visible and EPR spectroscopies. In D. gigas, higher amounts of Hmc can be obtained from the membranes than from the soluble fraction. This enabled further characterization of both cytochromes. The apparent heme reduction potentials of both Hmcs, determined at pH 7.5 through visible and EPR redox titrations, span a large range of redox potentials, approximately between 0 and –280?mV, and can be roughly divided into three groups: four to five hemes have E ⁰s of –30?mV to –100?mV, three to four hemes have E ⁰s around –170?mV, and seven to eight hemes have a lower E ⁰ of –250 to –280?mV. Several of these redox potentials are strongly pH dependent. Mössbauer studies of oxidized and reduced D. vulgaris Hmc show that this protein contains two high-spin hemes in both oxidation states. The rate of reduction of both Hmcs with the periplasmic hydrogenases from the corresponding organisms is extremely slow.     

Crystal structure of cytochrome c' from Rhodocyclus gelatinosus and comparison with other cytochromes c'

Cytochromes c' are heme proteins found in photosynthetic and denitrifying bacteria, where they are presumably involved in electron transport. The cytochrome c' isolated from the bacterium Rhodocyclus gelatinosus (RGCP) forms a homodimer with each polypeptide containing 129 residues. It has been crystallised in ammonium sulfate at pH?6. Crystals belong to space group P3₁21 with cell parameters a?=?70.2?Å and c?=?126.8?Å, which corresponds to a dimer in the asymmetric unit (VM?=?3.5?ų?/?Da). The crystal structure of RGCP was solved by the molecular replacement method and refined using data to 2.5-Å resolution. The final crystallographic R factor was 17.9% for all reflections (above 2?σ) in the resolution range 27.4 to 2.5?Å. The refined model includes 1876 non-hydrogen protein atoms and 56 water molecules. As typical of c–type cytochromes, the heme group is covalently bound to Cys-X-Y-Cys-His through thio-ether bonds, and His123 occupies the fifth axial coordination position. On the vacant "distal" site, Phe16 blocks the direct access to the sixth coordination site, which is in a predominantly hydrophobic environment. In spite of the low sequence homology among cytochromes c' the overall fold is similar. The monomer structure consists of 4 anti-parallel α-helices and has random coils in the loops between the helices, and at the N- and C-termini. The subunits cross each other to form an X shape.     

Isolation and characterization of two soluble heme c-containing proteins from Chromatium vinosum

The photosynthetic purple sulfur bacterium Chromatium vinosum has been shown to possess two previously undetected heme c-containing, soluble proteins. One is an acidic, c-type cytochrome with a molecular weight of 12 300 and an oxidation-reduction midpoint potential (at pH 8.0) of ?82 mV. The other protein is a basic protein with a molecular weight of 11 900 and an oxidation-reduction midpoint potential (at pH 8.0) of ?110 mV. The basic protein, in both oxidized and reduced forms, has optical spectra similar to those of myoglobin and the oxidized C. vinosum protein exhibits a high-spin heme EPR spectrum similar to that of metmyoglobin. Furthermore, the basic C. vinosum protein binds CO and O₂. The spectra of the CO and O₂ complexes show significant similarities with the respective myoglobin complexes. Possible functions for an O₂-binding protein in C. vinosum are discussed.     

A method for in situ characterization of b- and c-type cytochromes in Escherichia coli and in Complex III from beef heart mitochondria by combined spectrum deconvolution and potentiometric analysis

An analytical technique for the in situ characterization of b- and c-type cytochromes has been developed. From evaluation of the results of potentiometric measurements and spectrum deconvolutions, it was concluded that an integrated best-fit analysis of potentiometric and spectral data gave the most reliable results. In the total cytochrome b content of cytoplasmic membranes from aerobically grown Escherichia coli, four major components are distinguished with α-band maxima at 77 K of 555.7, 556.7, 558.6 and 563.5 nm, and midpoint potentials at pH 7.0 of 46, 174, ?75 and 187 mV, respectively. In addition, two very small contributions to the α-band spectrum at 547.0 and 560.2 nm, with midpoint potentials of 71 and 169 mV, respectively, have been distinguished. On the basis of their spectral properties they should be designated as a cytochrome c and a cytochrome b, respectively. In Complex III, isolated from beef heart mitochondria, five cytochromes are distinguished: cytochrome c₁ (Λ_m(25°C) = 553.5 nm; E′₀ = 238 mV) and four cytochromes bΛ_m(25°C) = 558.6, 561.2, 562.1, 566.1 nm and E′₀ = ?83, 26, 85, ?60 mV).     

Cytolytic Peptide Fragments of Cyt1Aa from Bacillus thuringiensis subsp. israelensis

Cyt1Aa is the major and most active component of the parasporal crystal of the Gram-positive soil entomopathogenic bacterium Bacillus thuringiensis subsp. israelensis. The Cyt1Aa protoxin exhibits some hemolytic and cytolytic activity. However, highly active 22–25 kDa toxins are obtained after proteolysis of Cyt1Aa from both the N- and the C-termini. As shown in this study, preliminary binding of the protoxin to polylamellary liposomes or partial denaturation of Cyt1Aa and further processing by several exogenous proteases yielded short 4.9–11.5 kDa cytolytic peptide fragments of Cyt1Aa. The shortest 51 amino acid peptide was obtained after pre-incubation of Cyt1Aa with SDS and proteolysis with proteinase K. This peptide was purified, identified as the Ile87–Asp137 fragment of Cyt1Aa and was shown to exhibit more than 30 % hemolysis of rabbit erythrocytes.     

Structural and kinetic studies of imidazole binding to two members of the cytochrome <Emphasis Type="Italic">c</Emphasis><Subscript>6</Subscript> family reveal an important role for a conserved heme pocket residue

The amino acid at position 51 in the cytochrome c ₆ family is responsible for modulating over 100 mV of heme midpoint redox potential. As part of the present work, the X-ray structure of the imidazole adduct of the photosynthetic cytochrome c ₆ Q51V variant from Phormidium laminosum has been determined. The structure reveals the axial Met ligand is dissociated from the heme iron but remains inside the heme pocket and the Ω-loop housing the Met ligand is stabilized through polar interactions with the imidazole and heme propionate-6. The latter is possible owing to a 180° rotation of both heme propionates upon imidazole binding. From equilibrium and kinetic studies, a Val residue at position 51 increases the stability of the Fe–S(Met) interaction and also affects the dynamics associated with imidazole binding. In this respect, the k _obs for imidazole binding to Arabidopsis thaliana cytochrome c _6A, which has a Val at the position equivalent to position 51 in photosynthetic cytochrome c ₆, was found to be independent of imidazole concentration, indicating that the binding process is limited by the Met dissociation rate constant (about 1 s⁻¹). For the cytochrome c ₆ Q51V variant, imidazole binding was suppressed in comparison with the wild-type protein and the V52Q variant of cytochrome c _6A was found to bind imidazole readily. We conclude that the residue type at position 51/52 in the cytochrome c ₆ family is additionally responsible for tuning the stability of the heme iron–Met bond and the dynamic properties of the ferric protein fold associated with endogenous ligand binding.     

Mechanisms of Mitochondrial Holocytochrome c Synthase and the Key Roles Played by Cysteines and Histidine of the Heme Attachment Site,Cys-XX-Cys-His

Mitochondrial cytochrome c assembly requires the covalent attachment of heme by thioether bonds between heme vinyl groups and a conserved CXXCH motif of cytochrome c/c₁. The enzyme holocytochrome c synthase (HCCS) binds heme and apocytochrome c substrate to catalyze this attachment, subsequently releasing holocytochrome c for proper folding to its native structure. We address mechanisms of assembly using a functional Escherichia coli recombinant system expressing human HCCS. Human cytochrome c variants with individual cysteine, histidine, double cysteine, and triple cysteine/histidine substitutions (of CXXCH) were co-purified with HCCS. Single and double mutants form a complex with HCCS but not the triple mutant. Resonance Raman and UV-visible spectroscopy support the proposal that heme puckering induced by both thioether bonds facilitate release of holocytochrome c from the complex. His-19 (of CXXCH) supplies the second axial ligand to heme in the complex, the first axial ligand was previously shown to be from HCCS residue His-154. Substitutions of His-19 in cytochrome c to seven other residues (Gly, Ala, Met, Arg, Lys, Cys, and Tyr) were used with various approaches to establish other roles played by His-19. Three roles for His-19 in HCCS-mediated assembly are suggested: (i) to provide the second axial ligand to the heme iron in preparation for covalent attachment; (ii) to spatially position the two cysteinyl sulfurs adjacent to the two heme vinyl groups for thioether formation; and (iii) to aid in release of the holocytochrome c from the HCCS active site. Only H19M is able to carry out these three roles, albeit at lower efficiencies than the natural His-19.     

Molecular Modeling of the Ternary Complex of Rusticyanin-Cytochrome c4-Cytochrome Oxidase: An Insight to Possible H-Bond Mediated Recognition and Electron Transfer Reaction in T.ferrooxidans

Abstract

The metabolism of Thiobacillus ferrooxidans involves electron transfer from the Fe⁺² ions in the extracellular environment to the terminal oxygen in the bacterial cytoplasm through a series of periplasmic proteins like Rusticyanin (RCy), Cytochrome (Cyt c₄), and Cytochrome oxidase (CcO). The energy minimization and MD studies reveal the stabilization of the three redox proteins in their ternary complex through the direct and water mediated H-bonds and electrostatic interaction. The surface exposed polar residues of the three proteins, i.e., RCy (His 143, Thr 146, Lys 81, Glu 20), Cyt c₄ (Asp 5, 15, 52, Ser 14, Glu 61), and CcO (Asp 135, Glu 126, 140, 142, Thr 177) formed the intermolecular hydrogen bonds and stabilized the ternary complex. The oxygen (O^εl) of Glu 126, 140, and 142 on subunit II of the CcO interact to the exposed side-chain and O^b atoms of the Asp 52 of Cyt c₄ and Glu 20 and Leu 12 of RCy. The Asp 135 of subunit II also forms H-bond with the N^ε atom of Lys 81 of RCy. The O^εl of Glu 61 of Cyt c₄ is also H-bonded to O^γ atom of Thr 177 of CcO. Solvation followed by MD studies of the ternary protein complex revealed the presence of seven water molecules in the interfacial region of the interacting proteins. Three of the seven water molecules (W 79, W 437, and W 606) bridged the three proteins by forming the hydrogen bonded network (with the distances ～ 2.10–2.95 Å) between the Lys 81 (RCy), Glu 61 (Cyt c₄), and Asp 135 (CcO). Another water molecule W 603 was H-bonded to Tyr 122 (CcO) and interconnected the Lys 81 (RCy) and Asp 135 (CcO) through the water molecules W 606 and W 437. The other two water molecules (W 21 and W 455) bridged the RCy to Cyt c₄ through H-bonds, whereas the remaining W 76 interconnected the His 53 (Cytc₄) to Glu 126 (CcO) with distances ～ 2.95–3.0 Å.     

The catalytic reaction of cytochrome c oxidase probed by in situ gas titrations and FTIR difference spectroscopy

Cytochrome c oxidase (CcO) is a transmembrane heme‑copper metalloenzyme that catalyzes the reduction of O₂ to H₂O at the reducing end of the respiratory electron transport chain. To understand this reaction, we followed the conversion of CcO from Rhodobacter sphaeroides between several active-ready and carbon monoxide-inhibited states via attenuated total reflection Fourier-transform infrared (ATR FTIR) difference spectroscopy. Utilizing a novel gas titration setup, we prepared the mixed-valence, CO-inhibited R₂CO state as well as the fully-reduced R₄ and R₄CO states and induced the “active ready” oxidized state O_H. These experiments are performed in the dark yielding FTIR difference spectra exclusively triggered by exposure to O₂, the natural substrate of CcO. Our data demonstrate that the presence of CO at heme a₃ does not impair the catalytic oxidation of CcO when the cycle starts from the fully-reduced states. Interestingly, when starting from the R₂CO state, the release of the CO ligand upon purging with inert gas yield a product that is indistinguishable from photolysis-induced states. The observed changes at heme a₃ in the catalytic binuclear center (BNC) result from the loss of CO and are unrelated to electronic excitation upon illumination. Based on our experiments, we re-evaluate the assignment of marker bands that appear in time-resolved photolysis and perfusion-induced experiments on CcO.