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1.
Inorg Chem ; 56(21): 13205-13213, 2017 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-29053273

RESUMO

Nitrite coordination to heme cofactors is a key step in the anaerobic production of the signaling molecule nitric oxide (NO). An ambidentate ligand, nitrite has the potential to coordinate via the N- (nitro) or O- (nitrito) atoms in a manner that can direct its reactivity. Distinguishing nitro vs nitrito coordination, along with the influence of the surrounding protein, is therefore of particular interest. In this study, we probed Fe(III) heme-nitrite coordination in Alcaligenes xylosoxidans cytochrome c' (AXCP), an NO carrier that excludes anions in its native state but that readily binds nitrite (Kd ∼ 0.5 mM) following a distal Leu16 → Gly mutation to remove distal steric constraints. Room-temperature resonance Raman spectra (407 nm excitation) identify ν(Fe-NO2), δ(ONO), and νs(NO2) nitrite ligand vibrations in solution. Illumination with 351 nm UV light results in photoconversion to {FeNO}6 and {FeNO}7 states, enabling FTIR measurements to distinguish νs(NO2) and νas(NO2) vibrations from differential spectra. Density functional theory calculations highlight the connections between heme environment, nitrite coordination mode, and vibrational properties and confirm that nitrite binds to L16G AXCP exclusively through the N atom. Efforts to obtain the nitrite complex crystal structure were hampered by photochemistry in the X-ray beam. Although low dose crystal structures could be modeled with a mixed nitrite (nitro)/H2O distal population, their photosensitivity and partial occupancy underscores the value of the vibrational approach. Overall, this study sheds light on steric determinants of heme-nitrite binding and provides vibrational benchmarks for future studies of heme protein nitrite reactions.


Assuntos
Citocromos c'/química , Nitritos/química , Alcaligenes , Complexos de Coordenação/química , Citocromos c'/genética , Citocromos c'/efeitos da radiação , Heme/química , Heme/efeitos da radiação , Ferro/química , Ferro/efeitos da radiação , Ligantes , Modelos Químicos , Estrutura Molecular , Nitritos/efeitos da radiação , Mutação Puntual , Espectroscopia de Infravermelho com Transformada de Fourier , Análise Espectral Raman
2.
Protein Sci ; 26(4): 737-748, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28097774

RESUMO

Thermophilic Hydrogenophilus thermoluteolus cytochrome c' (PHCP) exhibits higher thermal stability than a mesophilic counterpart, Allochromatium vinosum cytochrome c' (AVCP), which has a homo-dimeric structure and ligand-binding ability. To understand the thermal stability mechanism and ligand-binding ability of the thermally stable PHCP protein, the crystal structure of PHCP was first determined. It formed a homo-dimeric structure, the main chain root mean square deviation (rmsd) value between PHCP and AVCP being 0.65 Å. In the PHCP structure, six specific residues appeared to strengthen the heme-related and subunit-subunit interactions, which were not conserved in the AVCP structure. PHCP variants having altered subunit-subunit interactions were more severely destabilized than ones having altered heme-related interactions. The PHCP structure further revealed a ligand-binding channel and a penta-coordinated heme, as observed in the AVCP protein. A spectroscopic study clearly showed that some ligands were bound to the PHCP protein. It is concluded that the dimeric PHCP from the thermophile is effectively stabilized through heme-related and subunit-subunit interactions with conservation of the ligand-binding ability. BRIEF SUMMARY: We report the X-ray crystal structure of cytochrome c' (PHCP) from thermophilic Hydrogenophilus thermoluteolus. The high thermal stability of PHCP was attributed to heme-related and subunit-subunit interactions, which were confirmed by a mutagenesis study. The ligand-binding ability of PHCP was examined by spectrophotometry. PHCP acquired the thermal stability with conservation of the ligand-binding ability. This study furthers the understanding of the stability and function of cytochromes c.


Assuntos
Proteínas de Bactérias/química , Citocromos c'/química , Hydrogenophilaceae/enzimologia , Multimerização Proteica , Chromatiaceae/enzimologia , Cristalografia por Raios X , Estabilidade Enzimática , Temperatura Alta , Estrutura Quaternária de Proteína
3.
Protein Sci ; 26(3): 464-474, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27883268

RESUMO

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.


Assuntos
Proteínas de Bactérias/química , Monóxido de Carbono/química , Chromatiaceae/enzimologia , Citocromos c'/química , Cristalografia por Raios X , Domínios Proteicos , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína
4.
Adv Microb Physiol ; 67: 1-84, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26616515

RESUMO

Cytochromes c' are a group of class IIa cytochromes with pentacoordinate haem centres and are found in photosynthetic, denitrifying and methanotrophic bacteria. Their function remains unclear, although roles in nitric oxide (NO) trafficking during denitrification or in cellular defence against nitrosoative stress have been proposed. Cytochromes c' are typically dimeric with each c-type haem-containing monomer folding as a four-α-helix bundle. Their hydrophobic and crowded distal sites impose severe restrictions on the binding of distal ligands, including diatomic gases. By contrast, NO binds to the proximal haem face in a similar manner to that of the eukaryotic NO sensor, soluble guanylate cyclase and bacterial analogues. In this review, we focus on how structural features of cytochromes c' influence haem spectroscopy and reactivity with NO, CO and O2. We also discuss the relevance of cytochrome c' to understanding the mechanisms of gas binding to haem-based sensor proteins.


Assuntos
Bactérias/enzimologia , Monóxido de Carbono/metabolismo , Citocromos c'/química , Citocromos c'/metabolismo , Heme/metabolismo , Óxido Nítrico/metabolismo , Oxigênio/metabolismo , Citocromos c'/genética , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Análise Espectral
5.
J Biol Inorg Chem ; 20(6): 949-56, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26100643

RESUMO

Cytochromes c', that occur in methanotrophic, denitrifying and photosynthetic bacteria, form unusual proximal penta-coordinate NO complexes via a hexa-coordinate distal NO intermediate. Their NO binding properties are similar to those of the eukaryotic NO sensor, soluble guanylate cyclase, for which they provide a valuable structural model. Previous studies suggested that hydrogen bonding between the displaced proximal histidine (His120) ligand (following its dissociation from heme due to trans effects from the distally bound NO) and a conserved aspartate residue (Asp121) could play a key role in allowing proximal NO binding to occur. We have characterized three variants of Alcaligenes xylosoxidans cytochrome c' (AXCP) where Asp121 has been replaced by Ala, Ile and Gln, respectively. In all variants, hydrogen bonding between residue 121 and His120 is abolished yet 5-coordinate proximal NO species are still formed. Our data therefore demonstrate that the His120-Asp121 bond is not essential for proximal NO binding although it likely provides an energy minimum for the displaced His ligand. All variants have altered proximal pocket structure relative to native AXCP.


Assuntos
Citocromos c'/química , Histidina/química , Óxido Nítrico/química , Achromobacter denitrificans , Citocromos c'/ultraestrutura , Ligação de Hidrogênio , Ligantes , Modelos Moleculares
6.
Biochemistry ; 54(21): 3320-7, 2015 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-25961377

RESUMO

Five-coordinate heme nitrosyl complexes (5cNO) underpin biological heme-NO signal transduction. Bacterial cytochromes c' are some of the few structurally characterized 5cNO proteins, exhibiting a distal to proximal 5cNO transition of relevance to NO sensing. Establishing how 5cNO coordination (distal vs proximal) depends on the heme environment is important for understanding this process. Recent 5cNO crystal structures of Alcaligenes xylosoxidans cytochrome c' (AXCP) and Shewanella frigidimarina cytochrome c' (SFCP) show a basic residue (Arg124 and Lys126, respectively) near the proximal NO binding sites. Using resonance Raman (RR) spectroscopy, we show that structurally characterized 5cNO complexes of AXCP variants and SFCP exhibit a range of ν(NO) (1651-1671 cm(-1)) and ν(FeNO) (519-536 cm(-1)) vibrational frequencies, depending on the nature of the proximal heme pocket and the sample temperature. While the AXCP Arg124 residue appears to have little impact on 5cNO vibrations, the ν(NO) and ν(FeNO) frequencies of the R124K variant are consistent with (electrostatically) enhanced Fe(II) → (NO)π* backbonding. Notably, RR frequencies for SFCP and R124A AXCP are significantly displaced from the backbonding trendline, which in light of recent crystallographic data and density functional theory modeling may reflect changes in the Fe-N-O angle and/or extent of σ-donation from the NO(π*) to the Fe(II) (dz(2)) orbital. For R124A AXCP, correlation of vibrational and crystallographic data is complicated by distal and proximal 5cNO populations. Overall, this study highlights the complex structure-vibrational relationships of 5cNO proteins that allow RR spectra to distinguish 5cNO coordination in certain electrostatic and steric environments.


Assuntos
Alcaligenes/enzimologia , Citocromos c'/química , Heme/química , Óxido Nítrico/química , Shewanella/enzimologia , Análise Espectral Raman , Alcaligenes/química , Modelos Moleculares , Shewanella/química
7.
J Biol Inorg Chem ; 20(4): 675-86, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25792378

RESUMO

The cytochromes c' (CYTcp) are found in denitrifying, methanotrophic and photosynthetic bacteria. These proteins are able to form stable adducts with CO and NO but not with O2. The binding of NO to CYTcp currently provides the best structural model for the NO activation mechanism of soluble guanylate cyclase. Ligand binding in CYTcps has been shown to be highly dependent on residues in both the proximal and distal heme pockets. Group 1 CYTcps typically have a phenylalanine residue positioned close to the distal face of heme, while for group 2, this residue is typically leucine. We have structurally, spectroscopically and kinetically characterised the CYTcp from Shewanella frigidimarina (SFCP), a protein that has a distal phenylalanine residue and a lysine in the proximal pocket in place of the more common arginine. Each monomer of the SFCP dimer folds as a 4-alpha-helical bundle in a similar manner to CYTcps previously characterised. SFCP exhibits biphasic binding kinetics for both NO and CO as a result of the high level of steric hindrance from the aromatic side chain of residue Phe 16. The binding of distal ligands is thus controlled by the conformation of the phenylalanine ring. Only a proximal 5-coordinate NO adduct, confirmed by structural data, is observed with no detectable hexacoordinate distal NO adduct.


Assuntos
Monóxido de Carbono/química , Citocromos c'/química , Óxido Nítrico/química , Sítios de Ligação , Monóxido de Carbono/metabolismo , Citocromos c'/metabolismo , Conformação Molecular , Óxido Nítrico/metabolismo , Shewanella/enzimologia
8.
Photosynth Res ; 124(1): 19-29, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25519852

RESUMO

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(-1), 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'.


Assuntos
Chromatiaceae/metabolismo , Citocromos c'/química , Citocromos c'/metabolismo , Heme/metabolismo , Temperatura , Varredura Diferencial de Calorimetria , Cristalografia por Raios X , Proteínas Mutantes/metabolismo , Desnaturação Proteica , Estabilidade Proteica , Análise Espectral Raman , Relação Estrutura-Atividade
9.
Chem Biodivers ; 10(9): 1574-88, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24078591

RESUMO

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.


Assuntos
Citocromos c'/química , Simulação de Dinâmica Molecular , Alcaligenes/metabolismo , Sítios de Ligação , Citocromos c'/metabolismo , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Estrutura Terciária de Proteína
10.
Biosci Biotechnol Biochem ; 77(8): 1677-81, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23924718

RESUMO

Sequence analysis indicated that thermophilic Hydrogenophilus thermoluteolus cytochrome c' (PHCP) and its mesophilic homolog, Allochromatium vinosum cytochrome c' (AVCP), closely resemble each other in a phylogenetic tree of the cytochrome c' family, with 55% sequence identity. The denaturation temperature of PHCP was 87 °C, 35 °C higher than that of AVCP. Furthermore, PHCP exhibited a larger enthalpy change value during its thermal denaturation than AVCP. While AVCP was dimeric, as observed previously, PHCP was trimeric, and this was the first observation as a cytochrome c'. Dissociation of trimeric PHCP and its protein denaturation reversibly occurred at the same time in a two-state transition manner. Therefore, PHCP is enthalpically more stable than AVCP, perhaps due to its unique trimeric form, in addition to the lower number of Gly residues in its putative α-helical regions.


Assuntos
Chromatiaceae/enzimologia , Citocromos c'/química , Estabilidade Enzimática , Hydrogenophilaceae/enzimologia , Sequência de Aminoácidos , Temperatura Alta , Filogenia , Desnaturação Proteica , Termodinâmica
11.
Biochemistry ; 51(33): 6556-67, 2012 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-22827326

RESUMO

The thermodynamic and spectroscopic properties of two soluble electron transport proteins, cytochrome (Cyt) c' and flavocytochrome c, isolated from thermophilic purple sulfur bacterium Thermochromatium (Tch.) tepidum were examined and compared with those of the corresponding proteins from a closely related mesophilic bacterium Allochromatium (Alc.) vinosum. These proteins share sequence identities of 82% for the cytochromes c' and 86% for the flavocytochromes c. Crystal structures of the two proteins have been determined at high resolutions. Differential scanning calorimetry and denaturing experiments show that both proteins from Tch. tepidum are thermally and structurally much more stable than their mesophilic counterparts. The denaturation temperature of Tch. tepidum Cyt c' was 22 °C higher than that of Alc. vinosum Cyt c', and the midpoints of denaturation using guanidine hydrochloride were 2.0 and 1.2 M for the Tch. tepidum and Alc. vinosum flavocytochromes c, respectively. The enhanced stabilities can be interpreted on the basis of the structural and sequence information obtained in this study: increased number of hydrogen bonds formed between main chain nitrogen and oxygen atoms, more compact structures and reduced number of glycine residues. Many residues with large side chains in Alc. vinosum Cyt c' are substituted by alanines in Tch. tepidum Cyt c'. Both proteins from Tch. tepidum exhibit high structural similarities to their counterparts from Alc. vinosum, and the different residues between the corresponding proteins are mainly located on the surface and exposed to the solvent. Water molecules are found in the heme vicinity of Tch. tepidum Cyt c' and form hydrogen bonds with the heme ligand and C-terminal charged residues. Similar bound waters are also found in the vicinity of one heme group in the diheme subunit of Tch. tepidum flavocytochrome c. Electron density map of the Tch. tepidum flavocytochrome c clearly revealed the presence of disulfur atoms positioned between two cysteine residues at the active site near the FAD prosthetic group. The result strongly suggests that flavocytochrome c is involved in the sulfide oxidation in vivo. Detailed discussion is given on the relationships between the crystal structures and the spectroscopic properties observed for these proteins.


Assuntos
Grupo dos Citocromos c/química , Citocromos c'/química , Flavoproteínas/química , Oxirredutases/química , Sequência de Aminoácidos , Varredura Diferencial de Calorimetria , Chromatiaceae/química , Cristalização , Cristalografia por Raios X , Modelos Moleculares , Estabilidade Proteica , Alinhamento de Sequência , Termodinâmica
12.
Antioxid Redox Signal ; 17(9): 1246-63, 2012 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-22356101

RESUMO

SIGNIFICANCE: Ligand selectivity for dioxygen (O(2)), carbon monoxide (CO), and nitric oxide (NO) is critical for signal transduction and is tailored specifically for each heme-protein sensor. Key NO sensors, such as soluble guanylyl cyclase (sGC), specifically recognized low levels of NO and achieve a total O(2) exclusion. Several mechanisms have been proposed to explain the O(2) insensitivity, including lack of a hydrogen bond donor and negative electrostatic fields to selectively destabilize bound O(2), distal steric hindrance of all bound ligands to the heme iron, and restriction of in-plane movements of the iron atom. RECENT ADVANCES: Crystallographic structures of the gas sensors, Thermoanaerobacter tengcongensis heme-nitric oxide/oxygen-binding domain (Tt H-NOX(1)) or Nostoc puntiforme (Ns) H-NOX, and measurements of O(2) binding to site-specific mutants of Tt H-NOX and the truncated ß subunit of sGC suggest the need for a H-bonding donor to facilitate O(2) binding. CRITICAL ISSUES: However, the O(2) insensitivity of full length sGC with a site-specific replacement of isoleucine by a tyrosine on residue 145 and the very slow autooxidation of Ns H-NOX and cytochrome c' suggest that more complex mechanisms have evolved to exclude O(2) but retain high affinity NO binding. A combined graphical analysis of ligand binding data for libraries of heme sensors, globins, and model heme shows that the NO sensors dramatically inhibit the formation of six-coordinated NO, CO, and O(2) complexes by direct distal steric hindrance (cyt c'), proximal constraints of in-plane iron movement (sGC), or combinations of both following a sliding scale rule. High affinity NO binding in H-NOX proteins is achieved by multiple NO binding steps that produce a high affinity five-coordinate NO complex, a mechanism that also prevents NO dioxygenation. FUTURE DIRECTIONS: Knowledge advanced by further extensive test of this "sliding scale rule" hypothesis should be valuable in guiding novel designs for heme based sensors.


Assuntos
Citocromos c'/metabolismo , Guanilato Ciclase/metabolismo , Heme/metabolismo , Hemeproteínas/metabolismo , Oxigênio/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Citocromos c'/química , Guanilato Ciclase/química , Hemeproteínas/química , Receptores Citoplasmáticos e Nucleares/química , Guanilil Ciclase Solúvel
13.
Proc Natl Acad Sci U S A ; 108(38): 15780-5, 2011 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-21900609

RESUMO

Carbon monoxide (CO) is a product of haem metabolism and organisms must evolve strategies to prevent endogenous CO poisoning of haemoproteins. We show that energy costs associated with conformational changes play a key role in preventing irreversible CO binding. AxCYTcp is a member of a family of haem proteins that form stable 5c-NO and 6c-CO complexes but do not form O(2) complexes. Structure of the AxCYTcp-CO complex at 1.25 Å resolution shows that CO binds in two conformations moderated by the extent of displacement of the distal residue Leu16 toward the haem 7-propionate. The presence of two CO conformations is confirmed by cryogenic resonance Raman data. The preferred linear Fe-C-O arrangement (170 ± 8°) is accompanied by a flip of the propionate from the distal to proximal face of the haem. In the second conformation, the Fe-C-O unit is bent (158 ± 8°) with no flip of propionate. The energetic cost of the CO-induced Leu-propionate movements is reflected in a 600 mV (57.9 kJ mol(-1)) decrease in haem potential, a value in good agreement with density functional theory calculations. Substitution of Leu by Ala or Gly (structures determined at 1.03 and 1.04 Å resolutions) resulted in a haem site that binds CO in the linear mode only and where no significant change in redox potential is observed. Remarkably, these variants were isolated as ferrous 6c-CO complexes, attributable to the observed eight orders of magnitude increase in affinity for CO, including an approximately 10,000-fold decrease in the rate of dissociation. These new findings have wide implications for preventing CO poisoning of gas-binding haem proteins.


Assuntos
Proteínas de Bactérias/química , Monóxido de Carbono/química , Citocromos c'/química , Conformação Proteica , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Monóxido de Carbono/metabolismo , Intoxicação por Monóxido de Carbono/metabolismo , Intoxicação por Monóxido de Carbono/prevenção & controle , Cristalização , Cristalografia por Raios X , Citocromos c'/genética , Citocromos c'/metabolismo , Compostos Ferrosos/química , Compostos Ferrosos/metabolismo , Heme/química , Heme/metabolismo , Humanos , Cinética , Modelos Químicos , Modelos Moleculares , Mutação , Oxirredução , Ligação Proteica , Análise Espectral Raman
14.
FEBS J ; 278(13): 2341-8, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21554540

RESUMO

Hydrogenophilus thermoluteolus cytochrome c' (PHCP) has typical spectral properties previously observed for other cytochromes c', which comprise Ambler's class II cytochromes c. The PHCP protein sequence (135 amino acids) deduced from the cloned gene is the most homologous (55% identity) to that of cytochrome c' from Allochromatium vinosum (AVCP). These findings indicate that PHCP forms a four-helix bundle structure, similar to AVCP. Strikingly, PHCP with a covalently bound heme was heterologously synthesized in the periplasm of Escherichia coli strains deficient in the DsbD protein, a component of the System I cytochrome c biogenesis machinery. The heterologous synthesis of PHCP by aerobically growing E. coli also occurred without a plasmid carrying the genes for Ccm proteins, other components of the System I machinery. Unlike Ambler's class I general cytochromes c, the synthesis of PHCP is not dependent on the System I machinery and exhibits similarity to that of E. coli periplasmic cytochrome b(562), a 106-residue four-helix bundle.


Assuntos
Chromatiaceae/metabolismo , Citocromos c'/metabolismo , Citocromos c/metabolismo , Escherichia coli/metabolismo , Heme/metabolismo , Hydrogenophilaceae/metabolismo , Periplasma/metabolismo , Sequência de Aminoácidos , Chromatiaceae/genética , Citocromos c/genética , Citocromos c/isolamento & purificação , Citocromos c'/genética , Citocromos c'/isolamento & purificação , Escherichia coli/genética , Hydrogenophilaceae/genética , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos
15.
J Phys Condens Matter ; 23(17): 176003, 2011 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-21493970

RESUMO

We perform Monte Carlo simulations in order to study the magnetic properties of the mixed spin-S = ± 3/2, ± 1/2 and spin-σ = ± 5/2, ± 3/2, ± 1/2 Ising model. The spins are alternated on a square lattice such that S and σ are nearest neighbors. We found that when the Hamiltonian includes antiferromagnetic interactions between the S and σ spins, ferromagnetic interactions between the spins S, and a crystal field, the system presents compensation temperatures in a certain range of the parameters. The compensation temperatures are temperatures below the critical point where the total magnetization is zero, and they have important technological applications. We calculate the finite-temperature phase diagrams of the system. We found that the existence of compensation temperatures depends on the strength of the ferromagnetic interaction between the S spins.


Assuntos
Biofísica/métodos , Magnetismo , Algoritmos , Citocromos c'/química , Ferro/química , Modelos Estatísticos , Método de Monte Carlo , Porfirinas/química , Teoria Quântica , Temperatura
16.
Biochemistry ; 50(6): 1029-41, 2011 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-21190388

RESUMO

To provide insight into the role of local sequence in the nonrandom coil behavior of the denatured state, we have extended our measurements of histidine-heme loop formation equilibria for cytochrome c' to 6 M guanidine hydrochloride. We observe that there is some reduction in the scatter about the best fit line of loop stability versus loop size data in 6 M versus 3 M guanidine hydrochloride, but the scatter is not eliminated. The scaling exponent, ν(3), of 2.5 ± 0.2 is also similar to that found previously in 3 M guanidine hydrochloride (2.6 ± 0.3). Rates of histidine-heme loop breakage in the denatured state of cytochrome c' show that some histidine-heme loops are significantly more persistent than others at both 3 and 6 M guanidine hydrochloride. Rates of histidine-heme loop formation more closely approximate random coil behavior. This observation indicates that heterogeneity in the denatured state ensemble results mainly from contact persistence. When mapped onto the structure of cytochrome c', the histidine-heme loops with slow breakage rates coincide with chain reversals between helices 1 and 2 and between helices 2 and 3. Molecular dynamics simulations of the unfolding of cytochrome c' at 498 K show that these reverse turns persist in the unfolded state. Thus, these portions of the primary structure of cytochrome c' set up the topology of cytochrome c' in the denatured state, predisposing the protein to fold efficiently to its native structure.


Assuntos
Proteínas de Bactérias/química , Citocromos c'/química , Rodopseudomonas/metabolismo , Guanidina/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína
17.
Anal Chem ; 83(3): 1015-21, 2011 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-21162592

RESUMO

Salts and buffers, commonly used in isolation and stabilization of biological analytes, have a deleterious effect on electrospray ionization mass spectrometry (ESI-MS). Excessive concentrations of salts lead to ion suppression and adduct formation, which mask or complicate ion signals. In this work, we describe a salt remover (SR), configured as a three-compartment flow-through system, where the central compartment is separated from the outer compartments by a cation-exchange membrane (CEM) and an anion-exchange membrane (AEM). One platinum electrode is placed in each of the outer compartments, where water or electrolyte is flowing. The CEM electrode is held at a negative potential relative to the AEM side; cations/anions migrate by electrophoresis to the CEM/AEM receiver liquids, respectively. Proteins have poorer electrophoretic mobility relative to the buffer components, permitting removal of the salt. The salt-free proteins proceed to the ESI source. The capillary scale SR (internal volume 2.5 µL) described here effectively desalted continuous flows of NaCl solutions (200 mequiv/L at 1 µL/min, equivalent to a flux of 200 nequiv/min with 88% efficiency) and achieved >99.8% salt removal with 154 mM NaCl (isotonic saline) at 1 µL/min. With optimized current, >80% of concurrently present 20 µM protein was transmitted. Desalting efficiency and analyte loss was evaluated with different salt concentration and flow rate combinations under different applied current. Good-quality ESI-MS spectra of cytochrome c, myoglobin, and lysozyme as test proteins in a saline solution, passed through the SR, are demonstrated.


Assuntos
Diálise/métodos , Sistemas On-Line , Cloreto de Sódio/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Bovinos , Galinhas , Citocromos c'/análise , Diálise/instrumentação , Eletrodos , Cavalos , Muramidase/análise , Mioglobina/análise , Sistemas On-Line/instrumentação , Espectrometria de Massas por Ionização por Electrospray/instrumentação
18.
J Phys Chem B ; 114(35): 11646-53, 2010 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-20704302

RESUMO

Understanding the dissociation mechanism of multimeric protein complex ions is important for deciphering gas phase dissociation experiments. The dissociation of cytochrome c' dimer ions in the gas phase was investigated in the present study by constrained molecular dynamics simulations. The center of mass (COM) distance between two monomers was selected as the constrained coordinate. The number of intermolecular hydrogen bonds, smallest distance of intermolecular residuals, value of dipole moments, root-mean-square deviations, and potential energy components of the force field as a function of COM distance were examined for different charge partitionings of the +10 total charge state. These data were rationalized with free energy profiles to produce a qualitative description of the dissociation process. When charges are symmetrically distributed between the monomers in the dimer, dissociation occurs at a well-defined distance with only small structural changes in the monomers. There is an elastic type of stretching that initially resists the separation of the monomers but after dissociation the monomers recoil slightly from this and relax. For asymmetrically distributed charges, the dissociation event is not nearly as well-defined because the more highly charged monomer unfolds before dissociation occurs. It is found in almost all cases, a charged N-terminus tethers this unfolding monomer to its dimer partner by binding in a nonspecific manner. This helps encourage monomer unfolding in the dissociation pathway. It is also shown that while the intermolecular Coulomb repulsion between the monomers is not the largest contribution to the overall potential energy, it dominates the potential energy difference between different charge states.


Assuntos
Citocromos c'/química , Gases/química , Dimerização , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Termodinâmica
19.
J Mol Microbiol Biotechnol ; 18(2): 102-8, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20185933

RESUMO

The cycP gene encoding a periplasmic cytochrome c' from the denitrifying beta-proteobacterium Achromobacter xylosoxidans was characterized. The genes flanking cycP encode components of a mobile genetic element characteristic of the beta-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.


Assuntos
Achromobacter denitrificans/enzimologia , Citocromos c'/biossíntese , Perfilação da Expressão Gênica , Achromobacter denitrificans/genética , Citocromos c'/genética , Citocromos c'/isolamento & purificação , Elementos de DNA Transponíveis , Escherichia coli/genética , Expressão Gênica , Periplasma/química , Sinais Direcionadores de Proteínas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação
20.
Eur Heart J ; 31(6): 728-36, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19933281

RESUMO

AIMS: We examined the impact of enterovirus (EV) cardiac replication activity on the endomyocardial mitochondrial pathway in patients with acute myocarditis. METHODS AND RESULTS: Levels of apoptotic cardiomyocytes were determined by TUNEL and ligation-mediated polymerase chain reaction (PCR) assays and EV replication activity was assessed by immunostaining of EV VP1 capsid protein in ventricular myocytes of patients with acute myocarditis (n = 25), and healthy heart controls (n = 15). Ratio of cytosolic/mitochondrial cytochrome c concentrations was determined by ELISA assay, levels of active caspase-9 were determined by western blot analysis and Bax/Bcl2 mRNA ratio was assessed by real-time reverse transcription-polymerase chain reaction (RT-PCR) in the same cardiac tissues. Patients with EV-associated acute myocarditis (n = 15) exhibited a significantly higher number of apoptotic cardiomyocytes than those with non-EV-associated acute myocarditis (n = 10) and controls (n = 15) (P < 0.001). Endomyocardial ratio of cytosolic/mitochondrial cytochrome c concentrations and levels of active caspase-9 protein were significantly increased in EV than in non-EV-related myocarditis patients (P < 0.001). Moreover, Bax/Bcl2 mRNA ratio was significantly increased in EV than in non-EV-related myocarditis patients (P < 0.001). CONCLUSION: Our findings evidence an EV-related activation of the cardiomyocyte mitochondrial apoptotic pathway in patients with acute myocarditis. Moreover, our results indicate that this EV-induced pro-apoptotic mechanism could be partly related to an up-regulation of Bax expression, and suggest that inhibition of this cell death process may constitute the basis for novel therapies.


Assuntos
Apoptose/fisiologia , Infecções por Enterovirus , Mitocôndrias Cardíacas/virologia , Miocardite/virologia , Miócitos Cardíacos/virologia , Adolescente , Adulto , Estudos de Casos e Controles , Caspase 9/metabolismo , Transformação Celular Viral , Citocromos c'/metabolismo , DNA Viral/análise , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Miocárdio/metabolismo , RNA Mensageiro/metabolismo , RNA Viral/análise , Proteínas Virais de Fusão/metabolismo , Adulto Jovem
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