RESUMO
Resonance Raman spectra of photolyzed carbonmonoxyhemoglobin obtained with 10-nanosecond pulses are compared with the spectra of photolyzed carbonmonoxyhemoglobin stabilized at 80 K. In comparing the deoxy with the photodissociated species, the changes in the Raman spectra are the same for these two experimental regimes. These results show that at ambient and cryogenic temperatures the heme pocket in liganded hemoglobin is significantly different from that of deoxyhemoglobin. It is concluded that measurements of the properties of intermediate species from photodissociated hemoglobin stabilized at low temperatures can be used to probe the short-lived metastable forms of hemoglobin present after photodissociation under biologically relevant solution conditions.
Assuntos
Hemoglobinas , Carboxihemoglobina , Fenômenos Químicos , Química , Congelamento , Humanos , Ligantes , Análise Espectral Raman , TemperaturaRESUMO
A dramatic change occurs in the vibrational properties of the iron-histidine bond, trans to the oxygen binding site, on freezing deoxyhemoglobin. The large, quaternary structure-dependent differences in the shape and frequency of the iron-histidine mode observed in resonance Raman scattering measurements above freezing ae significantly diminished by the freezing event and the scattering intensity increases substantially. On further reduction in temperature to 10 K this broad line becomes narrow and shifts to a higher frequency. These data implicate dynamical processes and protein interaction with water as contributors to the quaternary structure dependence of the iron-histidine bond and thus reflect on the role of that bond in the energetics of cooperative ligand binding.
Assuntos
Congelamento , Heme , Hemoglobina A , Regulação Alostérica , Histidina , Humanos , Movimento (Física) , Conformação Proteica , Análise Espectral Raman , ÁguaRESUMO
Picosecond time-resolved Raman spectra of hemoglobin generated with blue pulses (20 to 30 picoseconds) that were resonant with the Soret band and of sufficient intensity to completely photodissociate the starting liganded sample are reported. For both R- and T-state liganded hemoglobins, the peak frequencies in the spectrum of the deoxy transient were the same at approximately 25 picoseconds as those observed at 10 nanoseconds subsequent to photodissociation. In particular, the large R-T differences in the frequency of the stretching mode for the iron-proximal histidine bond (VFe-His) detected in previously reported nanosecond-resolved spectra were also evident in the picosecond-resolved spectra. The implications of this finding with respect to the distribution of strain energy in the liganded protein and the origin of the time course for geminate recombination are discussed. On the basis of these results, a microscopic model is proposed in which delocalization of strain energy is strongly coupled to the coordinate of the iron. The model is used to explain the origin of the R-T differences in the rates of ligand dissociation.
Assuntos
Hemoglobina A , Regulação Alostérica , Humanos , Movimento (Física) , Conformação Proteica , Análise Espectral Raman , Relação Estrutura-Atividade , TermodinâmicaRESUMO
Low-frequency resonance Raman spectra of transient hemoglobin species were observed within 10 nanoseconds of photolysis. The Raman frequencies of the iron-proximal histidine stretching mode for transient species having either the R or the T quaternary structure are higher than in the corresponding deoxy species. The observed frequency difference in the iron-histidine mode between the R- and T- state transients indicates that there are quaternary structure-dependent protein forces on the iron-histidine bond in the liganded hemoglobins. These differences are interpreted in terms of changes in the tilt of the histidine with respect to the heme plane.
Assuntos
Hemoglobinas , Carboxihemoglobina , Heme , Histidina , Humanos , Ferro , Movimento (Física) , Mioglobina , Fotólise , Análise Espectral RamanRESUMO
The picosecond geminate rebinding of molecular oxygen was monitored in a variety of different human, reptilian, and fish hemoglobins. The fast (100 to 200 picoseconds) component of the rebinding is highly sensitive to protein structure. Both proximal and distal perturbations of the heme affect this rebinding process. The rebinding yield for the fast process correlates with the frequency of the stretching motion of the iron-proximal histidine mode (VFe-His) observed in the transient Raman spectra of photodissociated ligated hemoglobins. The high-affinity R-state species exhibit the highest values for VFe-His and the highest yields for fast rebinding, whereas low affinity R-state species and T-state species exhibit lower values of VFe-His and correspondingly reduced yields for this geminate process. These findings link protein control of ligand binding with events at the heme.
Assuntos
Hemoglobinas Anormais/genética , Animais , Peixes , Heme/metabolismo , Hemoglobinas Anormais/metabolismo , Humanos , Ligantes/metabolismo , Oxigênio/metabolismo , Ligação Proteica , Conformação ProteicaRESUMO
The resonance Raman spectra of various species of spinach nitrite reductase (ferredoxin: nitrite oxidoreductase, EC 1.7.7.1) have been obtained with Soret excitation. These spectra allow for the vibrational properties of the unique siroheme chromophore at the enzyme's active site. The wholesale reordering of siroheme vibrational properties relative to those of protoporphyrins can be rationalized as resulting from a combination of symmetry lowering and bond order reductions within the siroheme macrocyle.
Assuntos
Heme/análogos & derivados , NADH NADPH Oxirredutases/análise , Nitrito Redutases/análise , Plantas/enzimologia , Sítios de Ligação , Heme/análise , Análise Espectral RamanRESUMO
Resonance Raman spectroscopy was employed to characterize the local heme environment of a high-spin, ligand-binding heme protein from Chromatium vinosum (Chromatium high-spin hemoprotein). High-frequency spectra obtained with both B- and Q-band excitation were found to resemble qualitatively those of deoxyhemoglobin (HbA). Differences between HbA and Chromatium high-spin hemoprotein spectra can be assigned to either the effects of a covalent linkage of the heme vinyls to the protein matrix or alterations in the heme-proximal ligand bonding interaction. Both kinematic and electronic effects were evident. The behavior of heme core-size sensitive modes and low-frequency modes in Chromatium high-spin hemoprotein may be an indication of distortions in the heme geometry of Chromatium high-spin hemoprotein relative to HbA. The effects of covalent bonding of the heme peripheral vinyls upon the vibrational, electronic, and geometric characteristics of the heme active site in Chromatium high-spin hemoprotein are discussed.
Assuntos
Chromatium/análise , Hemeproteínas , Oxigênio/metabolismo , Análise Espectral Raman , Hemeproteínas/metabolismo , Hemoglobina A , HemoglobinasRESUMO
Resonance Raman spectra of bc1 complexes from Rhodospirillum rubrum have been obtained. Various resonance conditions and the stoichiometric redox titration of the complex were used to isolate and identify the contributions of the heme c1 and heme b active sites to the observed spectra. The complex was found to partially photoreduce when exposed to laser excitation.
Assuntos
Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Rhodospirillum rubrum/análise , Análise Espectral Raman , Ácido Ascórbico/farmacologia , Ditionita/farmacologia , Eletroquímica , Transporte de Elétrons , Ferricianetos/farmacologia , Heme/metabolismo , Oxirredução , Fotoquímica , EspectrofotometriaRESUMO
Resonance Raman spectroscopy (RRS) has been employed to characterize cytochromes c1 isolated from bc1 complexes of beef heart mitochondria and Rhodopseudomonas sphaeroides. The data obtained in this study extend the physical characterization of cytochromes c1 and focus on the effects of the local protein environment on the heme active site. While the general characteristics of the cytochromes c1 are similar to those of smaller soluble cytochromes c, the behavior of several core-size and ligation-sensitive heme modes reveal that significant systematic differences exist between those species. These, most likely, result from changes in the heme axial-ligand interactions.
Assuntos
Citocromos c1/química , Mitocôndrias Cardíacas/enzimologia , Animais , Sítios de Ligação , Bovinos , Heme/química , Oxirredução , Rhodobacter sphaeroides/enzimologia , Análise Espectral RamanRESUMO
Two cytochromes have been isolated from chlorophyll-free bean sprouts, purified and characterized. The more abundant cytochrome was purified to apparent homogeneity and exhibits visible region absorbance maxima at 416, 520 and 550 nm in the reduced form and at 410 and 530 nm in the oxidized form. Although Resonance Raman spectra of this cytochrome closely resemble those of c-type cytochromes, pyridine hemochromogen analysis suggests that this cytochrome may contain a variant of heme c as its prosthetic group. The cytochrome has an apparent molecular mass of 12.5 kDa, an isoelectric point > 9.0 and a midpoint oxidation-reduction potential (Em) of -130 mV at pH 8.0. The less abundant of the two cytochromes, which was not completely purified, exhibits absorbance maxima at 438 and 560 nm in the reduced form and at 411 nm in the oxidized form and was shown to contain heme c as a prosthetic group. This cytochrome, which may also contain FAD, has an apparent molecular mass of approx. 38 kDa, an isoelectric point > 9.0 and Em = -300 mV. Preliminary results indicate that both cytochromes can form electrostatically-stabilized complexes with ferredoxin, suggesting the possibility that one or both of the cytochromes may participate in low-potential, non-photosynthetic electron transfer pathways involving ferredoxin.
Assuntos
Citocromos/isolamento & purificação , Fabaceae/enzimologia , Plantas Medicinais , Dicroísmo Circular , Citocromos/química , Citocromos/metabolismo , Ferredoxinas/metabolismo , Oxirredução , Espectrofotometria Ultravioleta , Análise Espectral RamanRESUMO
Resonance Raman spectra of cytochrome b6f complexes isolated from spinach chloroplasts have been obtained. Selective resonance enhancements and partial reductions of the complex by redox mediators were used to isolate and identify the contributions of heme b6 and heme f sites to the observed spectra. Corresponding spectra for turnip cytochrome f have also been obtained. Power-dependent photoreduction was observed in cytochrome f of the complex as well as in the isolated cytochrome f during the course of the Raman experiments.
Assuntos
Citocromos/química , Heme/química , Proteínas de Plantas/química , Citocromos f , Oxirredução , Análise Espectral RamanRESUMO
A recently discovered form of spinach catalase that contains both a novel heme and protoheme as prosthetic groups has been characterized using immunological and spectroscopic techniques. The enzyme appears to be a dimer of identical Mr 60,000 monomers. Extraction of the non-covalently bound prosthetic groups, followed by thin-layer chromatography of the extract, suggested that the novel heme contains four carboxylic acid side-chain groups. The resonance Raman spectrum of the resting enzyme indicates that the protoheme prosthetic group is five-coordinate and high-spin. The enzyme was shown to bind formate, azide and cyanide. Cyanide and azide binding to catalase are biphasic, suggesting the existence of two different binding sites for cyanide and azide in the enzyme. Results obtained from EPR and resonance Raman spectroscopies also support the hypothesis that two different ligand-binding sites are present in the enzyme. Western blots suggest that the Mr 60,000 peptide of the novel heme-containing catalase is similar or identical to that of a previously characterized, exclusively protoheme-containing, tetrameric catalase.
Assuntos
Catalase/metabolismo , Heme/análise , Plantas/enzimologia , Azidas/metabolismo , Sítios de Ligação , Western Blotting , Catalase/análise , Cianetos/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Eletroforese em Gel de Poliacrilamida , Formiatos/metabolismo , Substâncias Macromoleculares , Peso Molecular , Espectrofotometria , Análise Espectral Raman , VerdurasRESUMO
The results of a comprehensive Q-band resonance Raman investigation of cytochrome c1 and cytochrome f subunits of bc1 and b6f complexes are presented. Q-band excitation provides a particularly effective probe of the local heme environments of these species. The effects of protein conformation (particularly axial ligation) on heme structure and function were further investigated by comparison of spectra obtained from native subunits to those of a site directed c1 mutant (M183L) and various pH-dependent species of horse heart cytochrome c. In general, all species examined displayed variability in their axial amino acid ligation that suggests a good deal of flexibility in their hemepocket conformations. Surprisingly, the large scale protein rearrangements that accompany axial ligand replacement have little or no effect on macrocycle geometry in these species. This indicates the identity and/or conformation of the peptide linkage between the two cysteines that are covalently linked to the heme periphery may determine heme geometry.
Assuntos
Brassica/enzimologia , Citocromos c1/química , Citocromos/química , Rhodobacter capsulatus/enzimologia , Citocromos f , Complexo III da Cadeia de Transporte de Elétrons/química , Concentração de Íons de Hidrogênio , Mutagênese Sítio-Dirigida , Mutação , Análise Espectral RamanRESUMO
The interactions of ethanol and methanol with ferrihemoglobin were examined using resonance Raman spectroscopy. After binding either alcohol, the low-frequency resonance Raman spectra of human ferrihemoglobin are almost identical to the unperturbed spectrum except for shifts in the 309 cm-1 band to higher frequency by as much as 8 cm-1. The ethanol-induced shift is greater than that with methanol even though complex formation was less for ethanol than methanol. The spectral changes imply a site-specific, similar binding of these alcohols to ferrihemoglobin which may involve steric interactions. Possible assignments of the 309 cm-1 band to structural features as well as potential mechanisms of the alcohol-induced spectral changes are discussed.
Assuntos
Etanol/metabolismo , Metanol/metabolismo , Metemoglobina/metabolismo , Análise Espectral Raman , Animais , Sítios de Ligação , Cavalos , Humanos , Conformação ProteicaRESUMO
The focus of this study was to examine the functional role of the unusual peripheral substitution of heme A. The effects of heme A stereochemistry on the reconstitution of the porphyrin have been examined in the heme A-apo-myoglobin complex using optical absorption and resonance Raman and electron paramagnetic resonance spectroscopies. The addition of one equivalent of heme A to apo-Mb produces a complex which displays spectroscopic signals consistent with a distribution of high- and low-spin heme chromophores. These results indicate that the incorporation of heme A into apo-Mb significantly perturbs the protein refolding.
Assuntos
Heme/análogos & derivados , Mioglobina/química , Análise Espectral , Animais , Bovinos , Dimetil Sulfóxido , Espectroscopia de Ressonância de Spin Eletrônica , Compostos Férricos/química , Compostos Ferrosos/química , Heme/química , Conformação Proteica , Análise Espectral Raman , Relação Estrutura-AtividadeRESUMO
Resonance Raman spectra at cryogenic temperatures of photodissociated hemoglobins and the corresponding deoxygenated preparations are compared and significant differences are found in modes with contributions from peripheral substituents of the heme as well as in the iron-histidine stretching mode. These differences in heme vibrational spectra reflect differences in the tertiary structure of the heme pocket between deoxyhemoglobin and the CO-bound form. An analysis of the effects of cooperative energy storage on the tertiary structure around the heme is made and used to interpret this resonance Raman data. The differences between the spectra of the deoxygenated preparations and the photoproducts provide evidence that a fraction of the free energy of cooperativity, DeltaG, is located away from the heme. These data support models for cooperativity in which the cooperative energy is distributed over many bonds or is localized in protein bonds only, such as those at the subunit interface. In addition, the local changes in amino acid positions, which must occur following the change in the state of ligand binding, may drive the changes in the structural relationships of the subunits and hence form one of the initial steps for triggering the quaternary structure transition.
RESUMO
Time-resolved resonance Raman spectroscopy is a valuable tool for the study of the dynamics of heme-protein interactions. In particular, the photolysis of a ligand by short laser pulses allows for the examination of the dynamic evolution of heme-protein interactions subsequent to ligand dissociation. To date, such studies have been confined largely to hemoglobins and myoglobins. Here we present the results of the first transient Raman study of a peroxidase. Resonance Raman spectra of horseradish peroxidase were obtained within 10 ns of ligand (CO) photolysis at a variety of pH values. We find that there is only minimal relaxation of the heme pocket of horseradish peroxidase in response to ligand photolysis. This relaxation is pH-dependent and most probably involves the heme vinyl substituents. Such behavior is in sharp contrast to the transient behavior of most hemoglobins and beef heart cytochrome oxidase.