Unfolding and flexibility in hemoproteins shown in the case of carboxymethylated cytochrome c.

A circular dichroism study of carboxymethylated cytochrome c has been performed to obtain further information on the structural basis responsible for the observed changes in ligand binding and redox properties of the modified cytochrome c. The results give additional evidence of local structural changes occurring in the heme environment upon rupture of the (Met-80)-heme iron bond in the modified protein. This produces no alterations of the overall molecular conformation, but results in drastic changes in redox potential. In addition, analysis of the reversible conformational transitions induced by urea in the native and the modified proteins supports the idea that the modified derivative can be considered as an 'intermediate state' between the native and the fully unfolded protein.

Redox potentials of normal and SH(beta 93)-modified human hemoglobin. Effect of pH and D-glycerate 2,3-bisphosphate.

The effect of pH and D-glycerate 2,3-bisphosphate on the oxidation-reduction equilibria of human hemoglobin, normal and modified at the SH(beta 93) groups by reaction with cystine dimethylester has been studied with the aim of comparing the redox equilibria with the oxygen equilibria previously studied under the same experimental conditions. Analysis of data has shown that: (i) the same groups are involved in the oxygenation and oxidation alkaline Bohr effect; (ii) the pK values of these groups are lower in met-hemoglobin than in oxyhemoglobin; (iii) the difference in the affinity for D-glycerate 2,3-bisphosphate of ferric and deoxyhemoglobin is pH-independent in the normal protein, while significantly decreasing with a decrease in pH in the modified protein; (iv) the high cooperativity of the redox process (also occurring at acid pH) and the reduced oxidation Bohr effect observed in the modified protein are consistent with the destabilization of the T structure induced by the bulky reagent bound to the SH(beta 93) groups of the protein.

Aromatic amino-acids and subunit assembly in the hemoglobins from Scapharca inaequivalvis: a fluorescence and CD study of the apoproteins.

The dimeric and tetrameric hemoglobins from the mollusc Scapharca inaequivalvis have a unique assembly that places the heme-carrying E and F helices in the inside of the molecule. These helices form the intersubunit contact in the dimer, which represents the structural unit since the tetramer is a dimer of dimers. The E and F helices are highly conserved and contain about 70% of the phenylalanine and tyrosine residues, while the tryptophan residues are near the tetramer contact. The spectroscopic properties (circular dichroism and intrinsic fluorescence) of the aromatic amino-acid residues in the two globins indicate that heme removal brings about a larger conformational change in the tetrameric than in the dimeric protein and that the tryptophan residues acquire a more rigid conformation in the tetramer.

Differential in vitro translation of the precursors of bovine pancreatic trypsin inhibitor and its isoinhibitor II is controlled by the 5'-end region of their mRNAs.

Bovine spleen inhibitor (SI II), a 58-amino-acid protein present in several bovine tissues, is an isoinhibitor of bovine pancreatic trypsin inhibitor (BPTI or aprotinin). These two proteins, which differ in seven amino-acidic residues, have very similar inhibitory activity against serine proteinases and are biosynthesized as two separate precursors of 100 residues. Higher levels of BPTI, compared to SI II, are found in bovine lung, as well as in other bovine tissues, in contrast to the level in vivo of the corresponding mRNAs. SI mRNA possesses a 90-nt 5'-end region, absent in BPTI mRNA, with an additional 5' AUG in a different open reading frame (ORF). We have used an in vitro transcription/translation system to determine the effect of this upstream region on the efficiency of SI precursor translation. Full-length SI mRNA is translated in vitro 6-fold less efficiently than BPTI mRNA. However, when SI mRNA lacks the 5' non-coding region, the translational efficiency of the 'truncated' transcript is significantly increased, reaching the same level as that of BPTI mRNA. In all cases the 10,500 Da precursor is the product of the in vitro translation. Our results indicate that the dramatic differences in translational efficiency of the mRNAs encoding BPTI and SI II in vitro parallel the different levels of the two proteins in vivo, and could be attributed to the features of the 5' non-coding region of SI mRNA.

Direct electrochemical evidence for an equilibrium intermediate in the guanidine-induced unfolding of cytochrome c.

This paper reports a voltammetric and spectroscopic investigation of the guanidine-induced unfolding of cytochrome c at neutral pH and 25 degrees C. Electrochemical data provide direct evidence for the presence of an equilibrium intermediate (form I) strictly dependent on the denaturant concentration. The midpoint potential of form I has been determined (E1/2 = +0.010 V vs. NHE) and its structural features defined from analysis of the circular dichroism and absorbance spectroscopy data obtained under the same experimental conditions. From the correlation of electrochemical and spectroscopic data, we propose that the features detected by the intermediate conform to the molten globule state.

A circular dichroism study of the proton-linked transition in the carbomonoxy derivative of the hemoglobin component IV from trout.

Near ultraviolet and visible circular dichroism spectra of the carbomonoxy derivative of the hemoglobin component IV from trout Salmo irideus are pH-dependent in the range 6.2-7.8, and are affected by the presence of inositol hexaphosphate at pH 6.2. On the basis of previous studies, the spectral changes observed can be associated to the pH-dependent R4 leads to T4 transition occurring in the liganded protein. The CD spectra above 500 nm at low pH can be interpreted as due to release of the heme asymmetry in the T liganded conformation, in agreement with the movement of the iron toward the proximal histidine.

Oxidation reaction of Scapharca inaequivalvis hemoglobins with nitrite.

The oxidation reaction with nitrite of the dimeric and tetrameric hemoglobins from the mollusc Scapharca inaequivalvis has been studied kinetically and at equilibrium. In line with previous findings obtained with ferricyanide as oxidant, in both proteins the stable oxidation product is a hemichrome, although the nitrite-methemoglobin complex is formed in significant amount when excess nitrite is employed. The reaction kinetics are characterized by a lag period followed by an autocatalytic phase, as in the case of human hemoglobin. However, with respect to human hemoglobin, in the two molluscan proteins the lag phase is prolonged significantly due to the instability of their met-form, an obligatory intermediate for the onset of autocatalysis. All the data obtained in spectrophotometric, EPR and sedimentation velocity experiments under a variety of experimental conditions conform to the reaction mechanism proposed for human hemoglobin (Spagnuolo et al., Biochim. Biophys. Acta 911 (1987) 59-63) provided hemichrome formation and nitrite binding are taken into account.

Oxidation reaction of human oxyhemoglobin with nitrite: a reexamination.

The oxidation reaction of human oxyhemoglobin with nitrite is complex and is characterized by a lag period followed by an autocatalytic phase. On the basis of contradictory experimental results, in order to describe the time-course of the reaction, two different mechanisms have been proposed, involving either hydrogen peroxide or the superoxide anion as reaction intermediates. This paper reports a careful reinvestigation of this reaction carried out as a function of reagent concentration, buffer composition, presence of enzymatic scavengers of oxygen radicals or of other compounds which may affect the intermediate steps of the reaction. The results obtained show that: hydrogen peroxide can be definitely identified as the reaction intermediate, in agreement with the mechanism proposed by Kosaka et al. (Biochim. Biophys. Acta 702 (1982) 237-241); the reaction time-course depends in a different way on the concentrations of hemoglobin and nitrite, a finding that cannot be explained on the basis of this mechanism. A more complex reaction scheme is proposed, that provides a satisfactory description in quantitative terms for all the available experimental data.

CD studies on the reversed heme orientation in monomeric Glycera dibranchiata hemoglobins.

Circular dichroism spectra of three monomeric components of Glycera dibranchiata hemoglobins are reported. Contrary to what is found for most hemoglobins and myoglobins, G. dibranchiata hemoglobins display largely negative dichroic spectra in the Soret region. Independent NMR measurements have shown that the same monomeric hemoglobin components contain the heme moiety predominantly (greater than 85%) oriented in a reversed way with respect to the orientation which occurs in most hemoglobins and myoglobins. On the basis of these independent NMR studies, and also of previous data on other invertebrate hemoproteins, a correlation appears evident between reversed heme orientation in hemoglobins and negative ellipticity in the Soret CD spectrum. This represents a simple tool to evaluate this aspect of heme asymmetric environment.

Redox properties of components I and IV of trout hemoglobins: kinetic and potentiometric studies.

Redox properties of component I and IV from trout hemoglobin (Salmo irideus) have been studied kinetically and at equilibrium. In the case of component I of trout hemoglobin, the mid-point potential (Eh) is pH independent below the acid-alkaline transition (pKa approximately equal to 8.6) and decreases at higher pH, following the deprotonation of the water molecule. Similarly to human hemoglobin, the mid-point potential of component IV of trout hemoglobin is pH-dependent, but the redox Bohr effect is extended to more acid pH. Moreover, the cooperativity of the redox equilibrium process is higher than in human hemoglobin. These features parallel the oxygen-binding properties of the same hemoglobin components from trout hemolysate. Differently from human hemoglobin, the oxidation kinetics of the two hemoglobins from trout by potassium ferricyanide show markedly biphasic progress curves with pH-independent second-order rate constants. This behavior suggests a different energy barrier for the interaction with ferricyanide in the two types of subunit of both Hb components from trout.

Proteinase isoinhibitors from bovine spleen: primary structure of an intermediate in the processing of the precursor.

The complete amino acid sequence of the proteinase inhibitor III from bovine spleen is reported. It consists of 62 amino acid residues and is identical to that of spleen inhibitor II (an isoinhibitor of the bovine pancreatic trypsin inhibitor, which shares with the latter 89% of sequence identity), except for four extra residues at the C-terminal side. Inhibitor III appears to be an intermediate in the processing of the putative 100-residue primary expression product, which leads to the mature inhibitor II. These results and those previously obtained for another intermediate, isoinhibitor I, are indicative of the following order for the last steps of the precursor processing inhibitor I----inhibitor III----inhibitor II. The mature protein and the two intermediates isolated have a very similar antiproteolytic activity. However, their in vivo target enzyme(s) are not yet known, as also the target enzyme of the bovine pancreatic trypsin inhibitor is not known. Thus, the available data would indicate that either the three isoinhibitors have a distinct functional role, by inhibiting different target enzymes, or inhibitors I and III are obligatory intermediates for directing the final targeting of the mature, functionally relevant inhibitor II.

Localization and interaction of bovine pancreatic trypsin inhibitor and tryptase in the granules of bovine mast cells.

The interaction of bovine pancreatic trypsin inhibitor and bovine tryptase, isolated from liver capsule mast cells, was investigated. They form a complex in vitro with a Ki of 5.6 nM at pH 8.0 and are localized within the mast cell granules, as shown by immunogold staining at the electron microscope level. In addition, double immunogold electron microscopy revealed that the inhibitor and the enzyme are present in the same granules, where they occur in clusters; this may be taken as an indication of their interaction in vivo and suggests a physiological role for bovine pancreatic trypsin inhibitor in the regulation of tryptase proteolytic activity.

Studies on erythrocruorin. VII. Reconstitution of earthworm erythrocruorin from the apoprotein.

Apoerythrocruorin prepared from the giant respiratory hemoprotein of the earthworm (60 S, Mr = 3 X 10(-6)) is an electrophoretically homogeneous molecule which sediments as a single peak of low molecular weight (3.5 S) and has a lower alpha-helical content (approx. 30%) than the native protein. Titration of globin with ferric heme indicates the presence of different binding sites; however, after purification by ion exchange chromatography, the reconstitution product contains 1 haem/23 000 g of protein as the native molecule. Reconstituted ferric erythrocruorin is a low molecular weight hemichrome with the same optical and physicochemical properties of the hemichrome formed by natural ferric erythrocruorin. Reconstituted ferrous erythrocruorin reacquires the alpha-helical content and the quaternary structure of the native molecule. Reassociation into 10-S speices (1/12 of the whole molecules) is fast and easy, while that into whole molecules is slow and somewhat erratic. The functional properties of reconstituted ferrous erythrocruorin (oxygen affinity, cooperativity in oxygen binding, magnitude of Bohr effect) are very similar to those of the "stable" low cooperativity form of the undissociated protein.

Amphitrite ornata erythrocruorin. I. Structural properties and characterization of subunit interactions.

A high molecular weight erythrocruorin (Mr approx. 3 . 10(6)) is found in the vascular system of the marine terebellid worm Amphitrite ornata, while a low molecular weight hemoglobin is contained in the coelomic cells. Polyacrylamide gel electrophoresis indicates that Amphitrite erythrocruorin contains three different types of polypeptide chain, of molecular weight approx. 15,000, whereas the molecular weight per heme group is approx. 20,000. These data suggest that only two of three polypeptide chains may be associated with a heme group. The coelomic hemoglobin, which occurs as a monomer, has an apparent molecular weight of approx. 14,000. The circular dichroism spectra of Amphitrite erythrocruorin and of the coelomic protein reveal marked differences in the heme environment, while the alpha-helical contents are not very different (60% and 70%, respectively). Amphitrite erythrocruorin is unusual in its dissociation behavior. Divalent cations are required for maintaining the quaternary structure. In the pH range 7.75--8.5, when the Ca2+ concentration is reduced below 1 mM, the whole molecule (57 S) dissociates into a number of lower molecular weight species (25, 15, 10 and 3 S) which have been correlated with specific subunit structures by electron microscopy. Whole molecules and 25 S subunits are not in equilibrium with the lower molecular weight species and can be isolated from partially dissociated mixtures. In contrast, the lower molecular weight subunits are themselves in a state of rapid equilibrium which is sensitive to cations, protons and oxygen. Of special interest is the dimerization reaction of the 10 S subunits, which appears to be mediated by Ca2+ and conforms to the predictions of the Cann and Goad theory on ligand mediated equilibria. The dissociation of Amphitrite erythrocruorin is readily reversible when the Ca2+ concentration is increased. The subunits obtained at physiological (7.8) or slightly acid (6.5) pH completely reassemble into whole molecules. Reassembly, however, is only partial when dissociation occurs at high pH. The presence of stable intermediates, such as the 15 S species, may facilitate the reassociation process.

Infrared stretching frequencies of CO in carbomonoxyhemoglobin from trout.

The infrared spectra of the carbomonoxy derivatives of the hemoglobin components I and IV from trout have been measured in the CO stretching frequency region using a high resolution infrared spectrometer. The CO stretching frequency of Hb I CO is very close to that of carbomonoxy human hemoglobin and is pH-independent. In contrast, the CO stretching frequency of Hb IV CO is higher and shows a small but significant pH dependence in the range 6.2-7.8. These results point to a decreased strength of the iron-CO bond in Hb IV CO at low pH, in agreement with the conclusions drawn from the reported difference spectra of Hb IV CO as a function of pH.

Reconstitution of horse heart myoglobin with hemins methylated at 6- or 7-positions: a circular dichroism study.

The reconstitution kinetics of horse heart myoglobin, as met-cyano derivative, with two synthetic hemins in which the 6- or the 7-propionate is replaced by a methyl group, has been investigated by circular dichroism, in order to gain information on the heme re-orientation process following the heme insertion into the globin pocket. The results obtained confirm that the preferred heme orientation places the sole propionate into the position occupied by the 6-propionate in the crystal structure, supporting the importance of the salt bridge occurring between this propionate and the basic CD3 residue of the apoprotein. Moreover, they provide new information on the correlations existing between the shape and the intensity of the dichroic bands and the heme orientation inside the reconstituted protein. Our data suggest that positive Soret CD bands are associated with hemoglobins possessing, at equilibrium, the heme in the so-called 'correct' orientation (as in horse heart myoglobin), and negative dichroic bands are associated with hemoglobins possessing, at equilibrium, the heme in the so-called 'reversed' orientation (as for example, in Glycera dibranchiata hemoglobins). Thus, negligible contribution to the CD signal in reconstituted proteins can be associated to a 'wrong' orientation of the heme group, no matter whether the orientation at the equilibrium is the 'correct' or the 'reversed' one. Finally, the results obtained indicate that the perturbations due to the heme re-orientation appear as a local phenomenon, not affecting the distant domains of the macromolecule.

Proton-NMR investigation of the heme cavity in the cyanomet derivative of the cooperative homodimeric hemoglobin from Scapharca inaequivalvis.

The active-site structure of the paramagnetic cyanomet complex of the cooperative homodimeric hemoglobin from Scapharca inaequivalvis has been investigated by solution homonuclear NMR. In spite of the large size (32 kDa), the residues on the key proximal F- and distal E-helices could be sequence-specifically assigned and placed in the heme pocket in a manner common to diamagnetic systems. These backbone assignments were greatly facilitated by the significant dispersion of backbone chemical shifts by the highly anisotropic paramagnetic susceptibility tensor of the low-spin ferric state. The remainder of the residues in contact with the heme are assigned based on unique contacts to the heme predicted by the crystal structure and the observations of scalar connectivities diagnostic for the residues. The magnitude of the dipolar shifts for non-ligated residues was used to determine the anisotropy and orientation of the paramagnetic susceptibility tensor, and the major axis found tilted from the normal in a manner similar to that found for the Fe-CO unit in the crystal structure. The combination of NOESY inter-residue and heme-residue contacts, paramagnetic-induced relaxation and correlation between observed and dipolar shifts provide a description of the heme cavity in cyanomet Hb that is essentially the same as found in the carbonmonoxy Hb crystal structure. The pattern of both the heme methyl dominant contact shifts and the heme meso-proton dominant dipolar shifts are shown to be consistent with the orientation of the axial His. It is concluded that the present homonuclear NMR methods allow effective solution structure determination in the cyanomet form for dimeric Hb and suggest profitable extension to the tetrameric vertebrate hemoglobins.

Mini-myoglobin: native-like folding of the NO-derivative.

Mini-myoglobin is a polypeptide fragment (residues 32-139) obtained by limited proteolysis of horse heart apomyoglobin and reconstituted with the natural heme. Its functional properties are very similar to those of native myoglobin and therefore it may represent a model for testing the functional role of the protein fragment encoded by the central exon of myoglobin gene (residues 31-105). Here we have investigated some properties of the nitric oxide derivative of mini-myoglobin in comparison with those of NO-myoglobin, to provide more structural information on the heme pocket residues in addition to that already acquired by electron paramagnetic resonance of the cobalt-substituted mini-myoglobin. At pH 7.0, optical and circular dichroism Soret spectra, as well as electron paramagnetic resonance spectra reveal that the heme orientation in the pocket and the coordination state of the ferrous iron in NO-mini-myoglobin are similar to those of the whole protein. The spectra of the NO-mini-myoglobin complex are very sensitive to pH changes at variance to what is observed for the NO-myoglobin derivative in the same pH range (5.5-9.5). In particular, increasing or decreasing pH from 7.0, results in a decrease of the extinction coefficient and of the ellipticity in the Soret region and in a change of the shape of the electron paramagnetic resonance signal. The spectral changes are diagnostic for a transition from a hexa-coordinated (at pH 7.0) to a penta-coordinated heme (at pH 5.5 or 9.5), with the proximal histidine-iron bond either broken or stretched dramatically. Thus, although mini-myoglobin is able to bind NO in a geometry similar to that of the native protein, the resulting NO derivative shows a much higher pH dependence, suggesting that the two lacking side domains are mainly involved in enhancing the stability of the hemoprotein core.

Studies on Scapharca hemoglobins. Properties of the dimeric protein reconstituted with Fe- or Co-porphyrin.

A native globin from the dimeric hemoglobin, hemoglobin I, of the mollusc Scapharca inaequivalvis has been obtained with the acid-acetone method. The globin has a lower sedimentation coefficient than the native protein at neutral pH; its reconstitution product with natural heme has the same physicochemical and functional properties as the native protein. proto- and meso-cobalt hemoglobin I have been prepared and characterized. proto-Cobalt hemoglobin I binds oxygen reversibly with a lower affinity and a lower cooperativity than native hemoglobin I; thus, the changes in the functional properties brought about by substitution of iron with cobalt are similar to those observed in human hemoglobin A. The EPR spectra of deoxy-proto-cobalt hemoglobin I and of the photolysis product of oxy-meso-cobalt hemoglobin I indicate that two histidine residues are the apical heme ligands. The broad signal at g = 2.38 in deoxy-proto-cobalt hemoglobin I points to a constrained structure of the heme site in this derivative which results from a distorted coordination of the hindered proximal histidine. A similar structure has been proposed previously for the alpha chains in deoxy-cobalt hemoglobin A.

Physicochemical and functional properties of Perinereis cultrifera (Grübe) erythrocruorin.

Perinereis erythrocruorin has the following physicochemical properties: So20,w = 55S, corresponding to a molecular weight around 2.7-10(6); minimum molecular weight (on the basis of the heme content) 23 700 +/- 500; isoelectric point 5.1; alpha-helix content approximately 40%. At alkaline pH values in the oxygenated form the 55-S molecules dissociate into subunits with a weight average sedimentation coefficient of 3S, corresponding to a molecular weight approximately 35 000. Deoxygenation of partially dissociated samples promotes association of the 3-S subunits into a 9S component. The functional properties of Perinereis erythrocruorin are characterized by a low cooperativity in oxygen binding (n 1/2 = 1.5) at neutral pH. Cooperativity increases reversibly towards both the acid and alkaline pH range, irrespective of changes in molecular weight. This finding, taken together with the ultracentrifuge results, suggests that a subunit may represent the functional unit of the protein. The pH dependence of the oxygen affinity can be accounted for in terms of a single oxygen linked group with a pK of 8.