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Hydrogenophilus thermoluteolus, Thermochromatium tepidum , and Allochromatium vinosum , which grow optimally at 52, 49, and 25 °C, respectively, have homologous cytochromes c ′ (PHCP, TTCP, and AVCP, respectively) exhibiting at least 50% amino acid sequence identity. Here, the thermal stability of the recombinant TTCP protein was first confirmed to be between those of PHCP and AVCP. Structure comparison of the 3 proteins and a mutagenesis study on TTCP revealed that hydrogen bonds and hydrophobic interactions between the heme and amino acid residues were responsible for their stability differences. In addition, PHCP, TTCP, and AVCP and their variants with altered stability similarly bound nitric oxide and carbon oxide, but not oxygen. Therefore, the thermal stability of TTCP together with PHCP and AVCP can be tuned through specific interactions around the heme without affecting their gas-binding function. These cytochromes c ′ will be useful as specific gas sensor proteins exhibiting a wide thermal stability range. [ABSTRACT FROM AUTHOR]

Cytochrome c ′ is a nitric oxide (NO)-binding heme protein found in Gram negative bacteria. The thermal stability of psychrophilic Shewanella violacea cytochrome c ′ (SVCP) is lower than those of its homologues from other 2 psychrophilic Shewanella species, indicating that thermal destabilization mechanism for low-temperature adaptation accumulates in SVCP. In order to understand this mechanism at the amino acid level, here the stability and function of SVCP variants, modeled using the 2 homologues, were examined. The variants exhibited increased stability, and they bound NO similar to the wild type. The vulnerability as to the SVCP stability could be attributed to less hydrogen bond at the subunit interface, more flexible loop structure, and less salt bridge on the protein surface, which appear to be its destabilization mechanism. This study provides an example for controlling stability without spoiling function in psychrophilic proteins. [ABSTRACT FROM AUTHOR]

The dynamic structure changes, including the unfolding, dimerization, and transition from the compact to the open-bundle unfolding intermediate structure of Cyt c′, were detected by a small-angle neutron scattering experiment (SANS). The structure of Cyt c′ was changed into an unstructured random coil at pD = 1.7 (Rg = 25 Å for the Cyt c′ monomer). The four-α-helix bundle structure of Cyt c′ at neutral pH was transitioned to an open-bundle structure (at pD ~13), which is given by a numerical partial scattering function analysis as a joint-clubs model consisting of four clubs (α-helices) connected by short loops. The compactly folded structure of Cyt c′ (radius of gyration, Rg = 18 Å for the Cyt c′ dimer) at neutral or mildly alkaline pD transited to a remarkably larger open-bundle structure at pD ~13 (Rg = 25 Å for the Cyt c′ monomer). The open-bundle structure was also supported by ab initio modeling.

The stability of dimeric cytochrome c′ from a thermophile, as compared with that of a homologous mesophilic counterpart, is attributed to strengthened interactions around the heme and at the subunit-subunit interface, both of which are molecular interior regions. Here, we showed that interactions in the equivalent interior regions of homologous cytochromes c′ from two psychrophiles, Shewanella benthica and Shewanella violacea (SBCP and SVCP, respectively) were similarly weakened as compared with those of the counterparts of psychrophilic Shewanella livingstonensis and mesophilic Shewanella amazonensis (SLCP and SACP, respectively), and consistently the stability of SVCP, SLCP, and SACP increased in that order. Therefore, the stability of cytochromes c′ from the psychrophile, mesophile, and thermophile is systematically regulated in their molecular interior regions. Unexpectedly, however, the stability of SBCP was significantly higher than that of SVCP, and the former had additional molecular surface interactions. Collectively, SBCP had weakened interior interactions like SVCP did, but the former was stabilized at the molecular surface as compared with the latter, implying complex multiple adaptation of the proteins because the psychrophilic sources of SBCP and SVCP are also piezophilic, thriving in deep-sea extreme environments of low temperature and high hydrostatic pressure. [ABSTRACT FROM AUTHOR]

AVCP cytochromec′ from mesophilicAllochromatium vinosumexhibits lower stability than a thermophilic counterpart,Hydrogenophilus thermoluteoluscytochromec′ (PHCP), in which the six specific amino acid residues that are not conserved in AVCP are responsible for its stability. Here we measured the stability of AVCP variants carrying these specific residues instead of the original AVCP ones. Among the six single AVCP variants, all of which formed a dimeric structure similar to that of the wild-type, three were successfully stabilized compared with the wild-type, while one showed lower stability than the wild-type. In addition, the most stabilized and destabilized AVCP variants could bind CO, similar to the wild-type. These results indicated that mesophilic AVCP could be stabilized through specific three mutations modeled by the thermophilic counterpart, PHCP, without changing the CO binding ability. Mesophilic AVCP could be stabilized through mutations. [ABSTRACT FROM PUBLISHER]

The number of artificial protein supramolecules has been increasing; however, control of protein oligomer formation remains challenging. Cytochrome c′ from Allochromatium vinosum (AVCP) is a homodimeric protein in its native form, where its protomer exhibits a four-helix bundle structure containing a covalently bound five-coordinate heme as a gas binding site. AVCP exhibits a unique reversible dimer-monomer transition according to the absence and presence of CO. Herein, domain-swapped dimeric AVCP was constructed and utilized to form a tetramer and high-order oligomers. The X-ray crystal structure of oxidized tetrameric AVCP consisted of two monomer subunits and one domain-swapped dimer subunit, which exchanged the region containing helices αA and αB between protomers. The active site structures of the domain-swapped dimer subunit and monomer subunits in the tetramer were similar to those of the monomer subunits in the native dimer. The subunit-subunit interactions at the interfaces of the domain-swapped dimer and monomer subunits in the tetramer were also similar to the subunit-subunit interaction in the native dimer. Reduced tetrameric AVCP dissociated to a domain-swapped dimer and two monomers upon CO binding. Without monomers, the domain-swapped dimers formed tetramers, hexamers, and higher-order oligomers in the absence of CO, whereas the oligomers dissociated to domain-swapped dimers in the presence of CO, demonstrating that the domain-swapped dimer maintains the CO-induced subunit dissociation behavior of native ACVP. These results suggest that protein oligomer formation may be controlled by utilizing domain swapping for a dimer-monomer transition protein. [ABSTRACT FROM AUTHOR]

The article discusses a study aimed at understanding the protein stabilization mechanism of cytochrome c' (SACP) protein from mesophilic Shewanella amazonensis. The protein was evaluated to determine whether its thermal stability is intermediate between those of cytochrome c' (PHCP) from thermophilic Hydrogenophilus thermoluteolus and cytochrome c' (AVCP) from mesophilic Allochromatium vinosum. Results presented include protein purification, amino acid sequence, and three-spectral analysis.

A soluble cytochrome (Cyt) c' from thermophilic purple sulfur photosynthetic bacterium Thermochromatium ( Tch.) tepidum exhibits marked thermal tolerance compared with that from the closely related mesophilic counterpart Allochromatium vinosum. Here, we focused on the difference in the C-terminal region of the two Cyts c' and examined the effects of D131 and R129 mutations on the thermal stability and local heme environment of Cyt c' by differential scanning calorimetry (DSC) and resonance Raman (RR) spectroscopy. In the oxidized forms, D131K and D131G mutants exhibited denaturing temperatures significantly lower than that of the recombinant control Cyt c'. In contrast, R129K and R129A mutants denatured at nearly identical temperatures with the control Cyt c', indicating that the C-terminal D131 is an important residue maintaining the enhanced thermal stability of Tch. tepidum Cyt c'. The control Cyt c' and all of the mutants increased their thermal stability upon the reduction. Interestingly, D131K exhibited narrow DSC curves and unusual thermodynamic parameters in both redox states. The RR spectra of the control Cyt c' exhibited characteristic bands at 1,635 and 1,625 cm, ascribed to intermediate spin (IS) and high spin (HS) states, respectively. The IS/HS distribution was differently affected by the D131 and R129 mutations and pH changes. Furthermore, R129 mutants suggested the lowering of their redox potentials. These results strongly indicate that the D131 and R129 residues play significant roles in maintaining the thermal stability and modulating the local heme environment of Tch. tepidum Cyt c'. [ABSTRACT FROM AUTHOR]

The ability of hemoproteins to discriminate between diatomic molecules, and the subsequent affinity for their chosen ligand, is fundamental to the existence of life. These processes are often controlled by precise structural arrangements in proteins, with heme pocket residues driving reactivity and specificity. One such protein is cytochrome c', which has the ability to bind nitric oxide ( NO) and carbon monoxide ( CO) on opposite faces of the heme, a property that is shared with soluble guanylate cycle. Like soluble guanylate cyclase, cytochrome c' also excludes O2 completely from the binding pocket. Previous studies have shown that the NO binding mechanism is regulated by a proximal arginine residue ( R124) and a distal leucine residue ( L16). Here, we have investigated the roles of these residues in maintaining the affinity for NO in the heme binding environment by using various time-resolved spectroscopy techniques that span the entire femtosecond-second temporal range in the UV-vis spectrum, and the femtosecond-nanosecond range by IR spectroscopy. Our findings indicate that the tightly regulated NO rebinding events following excitation in wild-type cytochrome c' are affected in the R124 A variant. In the R124 A variant, vibrational and electronic changes extend continuously across all time scales (from fs-s), in contrast to wild-type cytochrome c' and the L16 A variant. Based on these findings, we propose a NO (re)binding mechanism for the R124 A variant of cytochrome c' that is distinct from that in wild-type cytochrome c'. In the wider context, these findings emphasize the importance of heme pocket architecture in maintaining the reactivity of hemoproteins towards their chosen ligand, and demonstrate the power of spectroscopic probes spanning a wide temporal range. [ABSTRACT FROM AUTHOR]

The article discusses the phylogenetics of hydrogenophilus thermoluteolus cytochrome c' (PHCP) and Allochromatium vinosum cytochrome c' (AVCP). It mentions that sequence analysis reveals matching of 55 percent sequence identities of PHCP and AVCP. It further states that large change in enthalpy of PHCP was observed during thermal denaturation and PHCP is enthalpically more stable than AVCP.

A comparative study of the early stages of unfolding of five proteins: cyt c, c-b 562, cyt c′, azurin, and lysozyme is reported. From crystallographic data, helical regions and intervening non-helical (or ‘turning’) regions are identified in each. Exploiting a previously introduced geometrical model, the paper describes quantitatively the stepwise extension of a polypeptide chain subject to the geometrical constraint that the spatial relationship among the residues of each triplet is fixed by native-state crystallographic data. Despite differences among the above-cited proteins, remarkable universality of behavior is found in the early stages of unfolding. At the very earliest stages, internal residues in each helical region have a common unfolding history; the terminal residues, however, are extraordinarily sensitive to structural perturbations. Residues in non-helical sections of the polypeptide unfold after residues in the internal helical regions, but with increasing steric perturbation playing a dominant role in advancing denaturation. [ABSTRACT FROM AUTHOR]

The cycP gene encoding a periplasmic cytochrome c′ from the denitrifying β-proteobacterium Achromobacter xylosoxidans was characterized. The genes flanking cycP encode components of a mobile genetic element characteristic of the β-proteobacteria, suggesting that cycP has inserted within a transposon or insertion element. The gene therefore does not form part of a denitrification operon or gene cluster. The level of expression of the cycP gene and the level of synthesis of its corresponding gene product were found to increase by maximally 3-fold anaerobically. Expression of cycP appears to occur mainly by non-specific read-through transcription from portions of the insertion element. Conditions were developed for high-level overproduction of cytochrome c′ in Escherichia coli, which resulted in signal peptide cleavage concomitant with secretion of the protein into the periplasm. Using a single-step purification, 20–30 mg of pure protein were isolated from a 1-litre culture. Based on UV-visible spectrophotometry the dimeric protein was shown to have a full complement of haem and to be indistinguishable from the native protein purified from A. xylosoxidans. This system provides an excellent platform to facilitate biochemical and structural dissection of the mechanism underlying the novel specificity of NO binding to the proximal face of the haem. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: The structure of the cytochrome c′ from the purple non-sulfur phototrophic bacterium Rubrivivax gelatinosus was determined using two crystals grown independently at pH 6.3 and pH 8. The resolution attained for the two structures (1.29Å and 1.50Å for the crystals at high and low pH, respectively) is the highest to date for this class of proteins. The two structures were compared in detail in an attempt to investigate the influence of pH on the geometry of the haem and of the coordination environment of the Fe(III) ion. However, while the results suggest some small propensity for the movement of the metal atom out of the plane of the haem ring upon pH increase, the accuracy of the measurements at these two pH below the pK of the axial histidine is not sufficient to provide hard evidence of a shift in the iron position and associated changes. [Copyright &y& Elsevier]

Abstract: Cytochromes-P460 of Nitrosomonas europaea and Methylococcus capsulatus (Bath), and the cytochrome c′ of M. capsulatus, believed to be involved in binding or transformation of N-oxides, are shown to represent an evolutionarily related new family of monoheme, ∼17kDa, cytochromes c found in the genomes of diverse Proteobacteria. All members of this family have a predicted secondary structure predominantly of beta-sheets in contrast to the predominantly alpha-helical cytochromes c′ found in photoheterotrophic and denitrifying Proteobacteria. [Copyright &y& Elsevier]

Absorption spectra of the purified cytochrome b6f complex from Chlamydomonas reinhardtii were monitored as a function of the redox potential. Four spectral and redox components were identified: in addition to heme f and the two b hemes, the fourth component must be the new heme ci (also denoted x) recently discovered in the crystallographic structures. This heme is covalently attached to the protein, but has no amino acid axial ligand. It is located in the plastoquinone-reducing site Qi in the immediate vicinity of a b hems. Each heme titrated as a one-electron Nernst curve, with midpoint potentials at pH 7.0 of -130 mV and -35 mV (hemes b), +100 my (heme ci, and +355 my (heme /). The reduced minus oxidized spectrum of heme ci consists of a broad absorption increase centered ≈425 nm. Its potential has a dependence of -60 mV/pH unit, implying that the reduced form binds one proton in the pH 6-9 range. The Qi site inhibitor 2-n-nonyl-4-hydroxyquinoline N-oxide, a semiquinone analogue, induces a shift of this potential by about -225 mV. The spectrum of ci matches the absorption changes previously observed in vivo for an unknown redox center denoted "G." The data are discussed with respect to the effect of the membrane potential on the electron transfer equilibrium between 6 and heme bH found in earlier experiments. [ABSTRACT FROM AUTHOR]

Abstract: Cytochrome c′ from the purple photosynthetic bacterium Allochromatium vinosum (CCP) displays a unique, reversible dimer-to-monomer transition upon binding of NO, CO, and CN−. This small, four helix bundle protein represents an attractive model for the study of other heme protein biosensors, provided a recombinant expression system is available. Here we report the development of an efficient expression system for CCP that makes use of a maltose binding protein fusion strategy to enhance periplasmic expression and allow easy purification by affinity chromatography. Coexpression of cytochrome c maturase genes and the use of a heme-rich Escherichia coli strain were found to be necessary to obtain reasonable yields of cytochrome c′. Characterization using circular dichroism, UV–vis spectroscopy, and size-exclusion chromatography confirms the native-like properties of the recombinant protein, including its ligand-induced monomerization. [Copyright &y& Elsevier]

Abstract: Simple MO arguments provide a qualitative explanation for the near-linear ON–Mn–NO arrangement observed for the trans-{Mn(NO)2}8 anion [Mn(Pc)(NO)2]−, which is unexpected for an Enemark–Feltham electron count n>6. The metal center in this species may be described as low-spin and the two unpaired electrons occupy a pair of eu orbitals composed of NO(π*) components, giving rise to a triplet ground state. In a certain sense, these eu SOMOs may be likened to the SOMO (singly occupied molecular orbital) of the allyl radical. The electronic structure of this species is quite different from that of diamagnetic dinitrosylheme intermediates, which have been spectroscopically characterized in synthetic studies as well as proposed for soluble guanylate cyclase and cytochrome c′. Some speculative remarks are offered as to why this proposal is not an unreasonable one from an electronic-structural perspective. [Copyright &y& Elsevier]

The dynamic structure changes, including the unfolding, dimerization, and transition from the compact to the open-bundle unfolding intermediate structure of Cyt c ', were detected by a small-angle neutron scattering experiment (SANS). The structure of Cyt c ' was changed into an unstructured random coil at pD = 1.7 (R g = 25 Å for the Cyt c ' monomer). The four-α-helix bundle structure of Cyt c ' at neutral pH was transitioned to an open-bundle structure (at pD ~13), which is given by a numerical partial scattering function analysis as a joint-clubs model consisting of four clubs (α-helices) connected by short loops. The compactly folded structure of Cyt c ' (radius of gyration, R g = 18 Å for the Cyt c ' dimer) at neutral or mildly alkaline pD transited to a remarkably larger open-bundle structure at pD ~13 (R g = 25 Å for the Cyt c ' monomer). The open-bundle structure was also supported by ab initio modeling.

Cytochrome c′ was isolated from the obligate methylotroph Methylococcus capsulatus Bath. The native and subunit molecular masses of the cytochrome were 34.9 kDa and 16.2 kDa, respectively, with an isoelectric pH of 7.0. The amino acid composition and N-terminal amino acid sequence were consistent with identification of the protein as a cytochrome c′. The electron paramagnetic resonance spectrum of the monoheme cytochrome indicated the presence of a high spin. S = 5/2, heme center that is diagnostic of cytochromes c′. The optical absorption spectra of ferric or ferrous cytochrome c′ were also characteristic of cytochromes c′, The ferrocytochrome bound carbon monoxide and nitric oxide, but not isocyanide, cyanide, or azide. Changes in physical properties due to binding of CO or NO to some other c′-type cytochromes have been interpreted as an indication of dimer dissociation. In the case of cytochrome c′ from M. capsulanes Bath, analytical ultracentrifugation of the ferricytochrome, the ferrocytochrome, and the ferrocytochrome-CO complex indicate that the changes induced by binding of CO are conformational and are not consistent with dimer dissociation. EPR spectra show that cytochrome c′ was reduced in the presence of hydroxylamine only when in a complex with cytochrome P-460. The value of the midpoint potential, Em 7.0 was -205 mV for cytochrome c′ from M. capsulatus Bath, which is well below the range of values reported for other cytochromes c′. The values of midpoint potentials for cytochrome P-460 (Em 7.0 = -300 mV to -380 mV) and cytochrome c555 (Em 7.0 = + 175 mV to + 195 mV) are less than and greater than, respectively, the value for cytochrome c′ and suggest the possibility that the latter may function as an electron shuttle between cytochrome P-460 and cytochrome c555. [ABSTRACT FROM AUTHOR]

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 Å3 / 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. [ABSTRACT FROM AUTHOR]

Three c-type cytochromes were purified from the filamentous sulfur-oxidizing bacterium, Beggiatoa alba strain B18LD, by ammonium sulfate fractionation, flat bed isoelectric focusing and gel filtration. Two of the cytochromes; flavocytochrome c-554 and cytochrome c′, were similar to cytochromes found in anoxygenic photosynthetic bacteria. Flavocytochrome c-554 had an apparent molecular weight of 21,000, an isoelectric focusing point at pH 4.4, contained FMN as the flavin component and had absorption maxima at 410, 450 and 470 nm in the oxidized form and at 417, 523 and 554 nm in the dithionite-reduced from. Cytochrome c′ was also an acidic protein with a pI of 4.8 and an apparent molecular weight of 18,000. The absorption spectra maxima were at 400, 490 and 635 nm in the oxidized form, at 424 and 550 nm in the dithione-reduced form and at 415 and 555 nm in the dithionite-reduced plus CO form. The third cytochrome characterized, cytochrome c-553 had an apparent molecular weight of 13,000, an isoelectric point at pH 4.4 and showed absorption maxima at 411 nm in the oxidized form and at 418, 523 and 553 nm in the dithionite-reduced form. Cytochrome c-553 was also isolated as a complex with a non-heme protein with a molecular weight of 16,000. The non-heme protein altered the absorption spectra and isoelectric point of cytochrome c-553. [ABSTRACT FROM AUTHOR]

The denitrifying bacterium, Achromobacter xylosoxidans NCIB 11015, was cultivated in meat extract-peptone medium and in Mn-free synthetic medium under denitrifying or non-denitrifying conditions. Electron paramagnetic resonance (EPR) spectra for the whole cells of the bacteria thus obtained were measured at 77K. The characteristic three-line signal was observed in the whole cells of the bacteria under denitrifying conditions, but not under non-denitrifying conditions. The three-line signal was more distinctly observed in the cells cultured in Mn-free medium. This signal could be assigned to nitrosylcytochrome c′ containing a five-coordinated nitrosylheme. The elemental composition in these cells is also discussed. [ABSTRACT FROM AUTHOR]

The number of artificial protein supramolecules has been increasing; however, control of protein oligomer formation remains challenging. Cytochrome c' from Allochromatium vinosum (AVCP) is a homodimeric protein in its native form, where its protomer exhibits a four-helix bundle structure containing a covalently bound five-coordinate heme as a gas binding site. AVCP exhibits a unique reversible dimer-monomer transition according to the absence and presence of CO. Herein, domain-swapped dimeric AVCP was constructed and utilized to form a tetramer and high-order oligomers. The X-ray crystal structure of oxidized tetrameric AVCP consisted of two monomer subunits and one domain-swapped dimer subunit, which exchanged the region containing helices αA and αB between protomers. The active site structures of the domain-swapped dimer subunit and monomer subunits in the tetramer were similar to those of the monomer subunits in the native dimer. The subunit-subunit interactions at the interfaces of the domain-swapped dimer and monomer subunits in the tetramer were also similar to the subunit-subunit interaction in the native dimer. Reduced tetrameric AVCP dissociated to a domain-swapped dimer and two monomers upon CO binding. Without monomers, the domain-swapped dimers formed tetramers, hexamers, and higher-order oligomers in the absence of CO, whereas the oligomers dissociated to domain-swapped dimers in the presence of CO, demonstrating that the domain-swapped dimer maintains the CO-induced subunit dissociation behavior of native ACVP. These results suggest that protein oligomer formation may be controlled by utilizing domain swapping for a dimer-monomer transition protein., (© 2016 The Protein Society.)

Cytochrome c' (SACP) from mesophilic Shewanella amazonensis, growing optimally at 37 °C, was thermally more stable than cytochrome c' (AVCP) from mesophilic Allochromatium vinosum, growing optimally at 25 °C. In contrast, SACP was less stable than cytochrome c' (PHCP) from thermophilic Hydrogenophilus thermoluteolus, growing optimally at 52 °C. Although only 28% of the SACP amino acid sequence was identical to those of AVCP and PHCP, the latter two being 55% identical, the overall main chain structures of the three cytochromes c' were similar, and SACP exhibited thermal stability intermediate between those of AVCP and PHCP. For these three proteins, the higher the stability is, the lesser the number of Gly residues in the putative α-helical regions is. Cytochromes c' including the present three are suitable for examining the protein stabilization mechanisms, because they are structurally similar and available from environments with a wide range of temperatures.

It is crucial to assign the correct redox and ligand states to crystal structures of proteins with an active redox centre to gain valid functional information and prevent the misinterpretation of structures. Single-crystal spectroscopies, particularly when applied in situ at macromolecular crystallography beamlines, allow spectroscopic investigations of redox and ligand states and the identification of reaction intermediates in protein crystals during the collection of structural data. Single-crystal resonance Raman spectroscopy was carried out in combination with macromolecular crystallography on Swiss Light Source beamline X10SA using cytochrome c' from Alcaligenes xylosoxidans. This allowed the fingerprinting and validation of different redox and ligand states, identification of vibrational modes and identification of intermediates together with monitoring of radiation-induced changes. This combined approach provides a powerful tool to obtain complementary data and correctly assign the true oxidation and ligand state(s) in redox-protein crystals.

Cytochrome c is a heme protein that is localized in the compartment between the inner and outer mitochondrial membranes where it functions to transfer electrons between complex III and complex IV of the respiratory chain. It can also form an intimate association with the mitochondrion-specific phospholipid cardiolipin that induces a conformational change in the protein enabling it to act as a peroxidase catalyzing the oxidation of cardiolipin and thereby instigating a chain of events that leads to apoptosis. Unlike the native protein, cytochrome c within the complex binds ligands rapidly; in particular, NO can coordinate to either the ferric or ferrous iron of the heme. Remarkably, in the ferrous form, NO binds preferentially to the proximal side of the heme and thus behaves in a way similar to cytochrome c'-type proteins and to guanylate cyclase. The implications of NO binding to the proapoptotic cytochrome c/cardiolipin complex are discussed in terms of modulating the apoptotic response and buffering NO concentrations. Insights into the structure of the complex are provided by comparison with cytochrome c' for which X-ray structures are available., (© 2014 Elsevier Inc. All rights reserved.)

Random-acceleration molecular-dynamics (RAMD) simulations with models of homodimeric 6-ligated distal-NO and 5-ligated proximal-NO cytochrome c' complexes, in TIP3 H2 O, showed two distinct, non-intercommunicating worlds. In the framework of a long cavity formed by four protein helices with heme at one extremity, NO was observed to follow different pathways with the two complexes to reach the solvent. With the 6-ligated complex, NO was observed to progress by exploiting protein internal channels created by thermal fluctuations, and be temporarily trapped into binding pockets before reaching the preferred gate at the heme end of the cavity. In contrast, with the 5-ligated complex, NO was observed to surface the solvent-exposed helix 7, up to a gate at the other extremity of the protein, only occasionally finding an earlier, direct way out toward the solvent. That only bulk NO gets involved in forming the 5-ligated proximal-NO complex is in agreement with previous experimental observations, while the occurrence of binding pockets suggests that also reservoir NO might play a role with the distal-NO complex., (Copyright © 2013 Verlag Helvetica Chimica Acta AG, Zürich.)