Spectroscopic characterization of heme A reconstituted myoglobin.

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.     

Reactivity of ferrous myoglobin at low pH.

The rates of reaction of myoglobin with carbon monoxide at low pH are reported. The pH versus rate profile of these kinetics resembles that found for heme model compounds, revealing an increase in combination rate at low pH. These facts suggest that CO binding by myoglobin changes from a mechanism of "direct ligant association" at pH 5 to a mechanism, similar to that proposed for heme model compounds, which assumes a tetracoordinated intermediate as a result of the protonation of the proximal imidazole.     

Alteration of sperm whale myoglobin heme axial ligation by site-directed mutagenesis

Three mutant proteins of sperm whale myoglobin (Mb) that exhibit altered axial ligations were constructed by site-directed mutagenesis of a synthetic gene for sperm whale myoglobin. Substitution of distal pocket residues, histidine E7 and valine E11, with tyrosine and glutamic acid generated His(E7)Tyr Mb and Val(E11)Glu Mb. The normal axial ligand residue, histidine F8, was also replaced with tyrosine, resulting in His(F8)Tyr Mb. These proteins are analogous in their substitutions to the naturally occurring hemoglobin M mutants (HbM). Tyrosine coordination to the ferric heme iron of His(E7)Tyr Mb and His(F8)Tyr Mb is suggested by optical absorption and EPR spectra and is verified by similarities to resonance Raman spectral bands assigned for iron-tyrosine proteins. His(E7)Tyr Mb is high-spin, six-coordinate with the ferric heme iron coordinated to the distal tyrosine and the proximal histidine, resembling Hb M Saskatoon [His(beta E7)Tyr], while the ferrous iron of this Mb mutant is high-spin, five-coordinate with ligation provided by the proximal histidine. His(F8)Tyr Mb is high-spin, five-coordinate in both the oxidized and reduced states, with the ferric heme iron liganded to the proximal tyrosine, resembling Hb M Iwate [His(alpha F8)Tyr] and Hb M Hyde Park [His(beta F8)Tyr]. Val(E11)Glu Mb is high-spin, six-coordinate with the ferric heme iron liganded to the F8 histidine. Glutamate coordination to the ferric iron of this mutant is strongly suggested by the optical and EPR spectral features, which are consistent with those observed for Hb M Milwaukee [Val(beta E11)Glu]. The ferrous iron of Val(E11)Glu Mb exhibits a five-coordinate structure with the F8 histidine-iron bond intact.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spectroscopic and electrochemical studies of horse myoglobin in dimethyl sulfoxide

This paper reports the first report of rapid, reversible direct electron transfer between a redox protein, specifically, horse myoglobin, and a solid electrode substrate in nonaqueous media and the spectroscopic (UV-vis, fluorescence, and resonance Raman) characterization of the relevant redox forms of myoglobin (Mb) in dimethyl sulfoxide (DMSO). In DMSO, the heme active site of metmyoglobin (metMb) appears to remain six-coordinate high-spin, binding water weakly. Changes in the UV-fluorescence spectra for metMb in DMSO indicate that the protein secondary structure has been perturbed and suggest that helix A has moved away from the heme. UV-vis and RR spectra for deoxyMb in DMSO suggest that the heme iron is six-coordinate low-spin, most likely coordinating DMSO. Addition of CO to deoxyMb in DMSO produces a single, photostable six-coordinate CO adduct. UV-vis and RR for Mb-CO in DMSO are consistent with a six-coordinate low-spin heme iron binding His93 weakly, if at all. The polarity of the distal heme pocket is comparable to that of the closed form of horse Mb-CO in aqueous solution, pH 7. Direct electron transfer between horse Mb and Au in DMSO solution was investigated by cyclic voltammetry. Mb exhibits stable and well-defined electrochemical responses that do not appear to be affected by the water content (1.3-7.5%). The electrochemical characteristics are consistent with a one-electron, quasi-reversible, diffusion-controlled charge transfer process at Au. E degrees for horse Mb in DMSO at Au is -0.241+/-0.005 V vs. NHE. The formal heterogeneous electron transfer rate constant, calculated from delta E(p) at 20 mV/s, is 1.7+/-0.5 x 10(-4) cm/s. The rate, which is unaffected by the presence of 1.3-7.5% water, is competitive with that previously reported for horse Mb in aqueous solution.     

Unfolding and pH studies on manganese peroxidase: role of heme and calcium on secondary structure stability

The present study characterizes the unfolding and folding processes of recombinant manganese peroxidase. This enzyme contains five disulfide bonds, two calcium ions, and one heme prosthetic group. Circular dichroism in the far UV was used to monitor global changes of the protein secondary structure, whereas UV-visible spectroscopy of the Soret band provided information about local changes in the heme cavity. The effects of reducing agents, oxidizing agents, and denaturants on this process were investigated. In addition to affecting the secondary structure content, these factors also affect the binding of the heme and the calcium ions, both of which have a significant effect on the folding process. Our results also show that denaturants induce irreversible changes, which are most likely due to the inability of the denatured protein to rebind either calcium or the heme. Breaking of disulfide bonds by 30 mM dithiothreitol causes complete unfolding of recombinant manganese peroxidase. The unfolding process was also studied at low and high pH, where the protein reaches the final unfolded state through two different intermediate states. The data also indicate that only the acidic folding-unfolding process is reversible. Our results indicate a complex synergistic relationship between the secondary structure content, the tertiary structure arrangement, and the binding of the heme and the calcium ions and disulfide bridge formation.     

Methanol-induced conformations of myoglobin at pH 4.0

The equilibrium methanol-induced conformation changes of holomyoglobin (hMb) at pH 4.0 have been studied by circular dichroism, tryptophan fluorescence, and Soret band absorption and by electrospray ionization mass spectrometry (ESI-MS). Optical spectra show the following: (1) In 35-40% (v/v) methanol/water, the native-like secondary structure remains, the tertiary structure is lost, the heme protein interactions are decreased, and a folding intermediate is formed. (2) In 50% methanol, heme is lost from the protein, and there is a small decrease in helicity together with a loss of tertiary structure. (3) At >60% methanol, the helicity increases and the apoprotein goes into a helical denatured state. The conformations are also probed by the charge states produced in ESI-MS and by hydrogen/deuterium (H/D) exchange with mass measurement by ESI-MS. At 0-30% methanol, native hMb produces relatively low charge states (9(+)-13(+)) in ESI-MS and exchanges relatively few hydrogens. In 35-40% methanol, at which an intermediate is formed, there is a bimodal distribution of hMb ions with both low (9(+)-13(+)) and high (14(+)-23(+)) charge states and also a high charge state distribution (12(+)-26(+)) of apomyoglobin (aMb) ions. Low and high charge states of hMb and a high charge state of aMb all show the same H/D exchange rate, indicating that an unfolded hMb intermediate interconverts between folded hMb and unfolded aMb. The charge state distribution for the unfolded hMb intermediate observed here is similar to that of the recently reported transient intermediate formed during the acid denaturation of hMb. At 50% alcohol the protein produces predominantly high charge states of aMb ions and shows H/D exchange rates close to those of the acid-denatured protein. H/D exchange of the helical denatured protein at alcohol concentrations >60%, at which high charge states of aMb are produced, shows that the protein structure is more protected than at approximately 50% methanol.     

Spectroscopic studies on heme d in the visible and infrared

Heme d has been isolated from the terminal oxidase complex of Escherichia coli strain MR43L/F152 and purified by high-pressure liquid chromatography. The infrared spectrum indicated that carbonyls in the chlorin skeleton of this isolated heme existed as carboxylic acids. Earlier work on the iron-free chlorin had demonstrated the presence of a spirolactone substituent. This may have arisen from a cyclization reaction from a dicarboxylic acid, diol precursor. Although the free heme in extracts can exist as a diol, this does not prove that the diol as such is the precise form in the enzyme complex. Visible and fluorescence spectra are reported for a variety of derivatives and complexes of heme d to establish a spectral library that may be used to prove the presence of this structure in other enzymes or cells. Association constants have been measured for complexes of heme d with cyanide, imidazole, and pyridine and are contrasted to available data for protoheme.     

Spectroscopic insights into axial ligation and active-site H-bonding in substrate-bound human heme oxygenase-2

Heme oxygenases (HOs) are monooxygenases that catalyze the first step in heme degradation, converting heme to biliverdin with concomitant release of Fe(II) and CO from the porphyrin macrocycle. Two heme oxygenase isoforms, HO-1 and HO-2, exist that differ in several ways, including a complete lack of Cys residues in HO-1 and the presence of three Cys residues as part of heme-regulatory motifs (HRMs) in HO-2. HRMs in other heme proteins are thought to directly bind heme, or to otherwise regulate protein stability or activity; however, it is not currently known how the HRMs exert these effects on HO-2 function. To better understand the properties of this vital enzyme and to elucidate possible roles of its HRMs, various forms of HO-2 possessing distinct alterations to the HRMs were prepared. In this study, variants with Cys265 in a thiol form are compared with those with this residue in an oxidized (part of a disulfide bond or existing as a sulfenate moiety) form. Absorption and magnetic circular dichroism spectroscopic data of these HO-2 variants clearly demonstrate that a new low-spin Fe(III) heme species characteristic of thiolate ligation is formed when Cys265 is reduced. Additionally, absorption, magnetic circular dichroism, and resonance Raman data collected at different temperatures reveal an intriguing temperature dependence of the iron spin state in the heme–HO-2 complex. These findings are consistent with the presence of a hydrogen-bonding network at the heme’s distal side within the active site of HO-2 with potentially significant differences from that observed in HO-1.     

Fluorescence decay kinetics of the tryptophyl residues of myoglobin: effect of heme ligation and evidence for discrete lifetime components

The fluorescence decay kinetics of the tryptophyl residues of sperm whale and yellowfin tuna myoglobin have been determined by using time-correlated single photon counting, with picosecond resolution. Purification by HPLC techniques resulted in the isolation of samples that exclusively displayed picosecond decay kinetics. Lifetimes of 24.4 ps for Trp14 and 122.0 ps for Trp7 were found for oxy sperm whale myoglobin (pH 7), which agree with theoretical predictions [Hochstrasser, R. M., & Negus, D. K. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 4399-4403]. The effects of ligand binding and pH on the decay kinetics were investigated, and the results were shown to be consistent with the known crystal structures. Data for the met form of sperm whale myoglobin were analyzed both in terms of a sum of discrete exponential components and as a continuous gamma distribution of exponential decays. The results were not found to support the existence of multiple, structurally distinct conformation states in myoglobin.     

X-ray absorption studies of myoglobin peroxide reveal functional differences between globins and heme enzymes

X-ray absorption studies of myoglobin peroxide show that although it is not identical with compound I or II of horseradish peroxidase [Chance, B., Powers, L., Ching, Y., Poulos, T., Yamazaki, I., & Paul, K. G. (1984) Arch. Biochem. Biophys. 235, 596-611], it has some structural features in common with both. As seen in compound I, the Fe-O distance is short, but the iron-pyrrole nitrogen distance is contracted with a longer iron-histidine distance like compound II. The iron has a higher oxidation state than Fe3+, suggesting an oxyferryl ion type species. Comparison of the structures of various peroxidase and myoglobin compounds points out systematic differences that may explain the catalytic activity of the pi cation radical as well as some of the differences between globins and heme enzymes.     

A primitive myoglobin from Tetrahymena pyriformis: its heme environment, autoxidizability, and genomic DNA structure

A myoglobin-like protein isolated from Tetrahymena pyriformis is composed of 121 amino acid residues. This is much smaller than sperm whale myoglobin by 32 residues, suggesting a distinct origin from the common globin gene. We have therefore examined this unique protein for its structural, spectral and stability properties. As a result, the rate of autoxidation of Tetrahymena oxymyoglobin (MbO(2)) was found to be almost comparable to that of sperm whale MbO(2) over a wide range of pH 4-12 in 0.1 M buffer at 25 degrees C. Moreover, both pH profiles exhibited the remarkable proton-assisted process, which can be performed in sperm whale myoglobin by the distal (E7) histidine as its catalytic residue. These kinetic observations are also in full accord with spectral examinations for the presence of a distal histidine in ciliated protozoa myoglobin. At the same time, we have isolated the globin genes both from T. pyriformis and Tetrahymena thermophila, and found that there is no intron in their genomic structures. This is in sharp contrast to previous reports on the homologous globin genes from Paramecium caudatum and Chlamydomonas eugametos. Rather, the Tetrahymena genes seemed to be related to the cyanobacterial globin gene from Nostoc commune. These contracted or truncated globins thus have a marked diversity in the cDNA, protein, and genomic structures.     

Coordination structure of the ferric heme iron in engineered distal histidine myoglobin mutants.

Recombinant human myoglobin mutants with the distal His residue (E7, His64) replaced by Leu, Val, or Gln residues were prepared by site-directed mutagenesis and expression in Escherichia coli. Electronic and coordination structures of the ferric heme iron in the recombinant myoglobin proteins were examined by optical absorption, EPR, 1H NMR, magnetic circular dichroism, and x-ray spectroscopy. Mutations, His-->Val and His-->Leu, remove the heme-bound water molecule resulting in a five-coordinate heme iron at neutral pH, while the heme-bound water molecule appears to be retained in the engineered myoglobin with His-->Gln substitution as in the wild-type protein. The distal Val and distal Leu ferric myoglobin mutants at neutral pH exhibited EPR spectra with g perpendicular values smaller than 6, which could be interpreted as an admixture of intermediate (S = 3/2) and high (S = 5/2) spin states. At alkaline pH, the distal Gln mutant is in the same so-called "hydroxy low spin" form as the wild-type protein, while the distal Leu and distal Val mutants are in high spin states. The ligand binding properties of these recombinant myoglobin proteins were studied by measurements of azide equilibrium and cyanide binding. The distal Leu and distal Val mutants exhibited diminished azide affinity and extremely slow cyanide binding, while the distal Gln mutant showed azide affinity and cyanide association rate constants similar to those of the wild-type protein.     

Neutron scattering studies of nucleosome structure at low ionic strength

Ionic strength studies using homogeneous preparations of chicken erythrocyte nucleosomes containing either 146 or 175 base pairs of DNA show a single unfolding transition at about 1.5 mM ionic strength as determined by small-angle neutron scattering. The transition seen by some investigators at between 2.9 and 7.5 mM ionic strength is not observed by small-angle neutron scattering in either type of nucleosome particle. The two contrasts measured (H2O and D2O) indicate that only small conformational changes occur in the protein core, but the DNA is partially unfolded below the transition point. Patterson inversion of the data and analysis of models indicate that the DNA in both types of particle is unwinding from the ends, leaving about one turn of supercoiled DNA bound to the histone core in approximately its normal (compact) conformation. The mechanism of unfolding appears to be similar for both types of particles and in both cases occurs at the same ionic strength. The unfolding observed for nucleosomes in this study is in definite disagreement with extended superhelical models for the DNA and also disagrees with models incorporating an unfolded histone core.     

Effect of pH on the iso-1-cytochrome c denatured state: changing constraints due to heme ligation

The effect of pH on the denatured state (3 M guanidine hydrochloride) was evaluated with fluorescence spectroscopy for four variants of iso-1-cytochrome c, AcTM (no surface histidines), AcH26 (surface histidine at position 26), AcH54 (surface histidine at position 54), and AcH54I52 (stabilizing I52 mutation added to AcH54). Changes in the compactness and the heme ligation of the denatured state, as a function of pH, were monitored through changes in Trp 59-heme fluorescence quenching. With the AcTM and AcH26 variants, no change in the fluorescence intensity occurs from pH 4 to 10. However, for the AcH54 and AcH54I52 variants the fluorescence intensity drops significantly between pH 4 and 6, consistent with His 54 binding to the heme of cytochrome c. Between pH 8 and 10 fluorescence intensity increases again, indicating that the His 54 is displaced from the heme. The data are consistent with lysines 4 and 5 being the primary heme ligands at alkaline pH, under denaturing conditions. This conclusion was confirmed by site-directed mutagenesis. Thermodynamic analysis indicates that heme-ligand affinity in the denatured state is controlled primarily by sequence position (loop size) and that when histidines are present they inhibit lysine ligation until approximately pH 8.5-9.0 as compared to pH 7.5 with the AcTM variant. Thus, at physiological pH, histidine ligands provide the primary constraint on the denatured state of cytochrome c. The heme-Trp 59 distance in the denatured state of iso-1-cytochrome c, derived from analysis by F?rster energy transfer theory, is approximately 26 A at pH 4 and 10, much shorter than the random coil prediction of 56 A. Surprisingly, the heme-Trp 59 distance in the His 54 bound conformation only drops to approximately 21 A, consistent with an extended conformation for the short polypeptide segment separating heme and Trp 59.     

Heme symmetry, vibronic structure, and dynamics in heme proteins: ferrous nicotinate horse myoglobin and soybean leghemoglobin

We report the visible and Soret absorption bands, down to cryogenic temperatures, of the ferrous nicotinate adducts of native and deuteroheme reconstituted horse heart myoglobin in comparison with soybean leghemoglobin-a. The band profile in the visible region is analyzed in terms of vibronic coupling of the heme normal modes to the electronic transition in the framework of the Herzberg-Teller approximation. This theoretical approach makes use of the crude Born-Oppenheimer states and therefore neglects the mixing between electronic and vibrational coordinates; however, it takes into account the vibronic nature of the visible absorption bands and allows an estimate of the vibronic side bands for both Condon and non-Condon vibrational modes. In this framework, an x-y splitting of the Q transition for native and deuteroheme reconstituted horse myoglobin is clearly assessed and attributed to electronic perturbations that, in turn, are caused by a reduction of the typical D(4h) symmetry of the system due to heme distortions of B(1g)-type symmetry and/or to an x-y asymmetric position of the nicotinate ring; in deuteroheme reconstituted horse myoglobin the asymmetric heme peripheral substituents add to the above effect(s). On the contrary, in leghemoglobin-a no spectral splitting upon nicotinate binding is observed, pointing to a planar heme configuration in which only distortions of A(1g)-type symmetry are effective and to which the nicotinate ring is bound in an x - y symmetric position. The local dynamic properties of the heme pocket of the three proteins are investigated through the temperature dependence of spectral line broadening. Leghemoglobin-a behaves as a softer matrix with respect to horse myoglobin, thus validating the hypothesis of a looser heme pocket conformation in the former protein, which allows a nondistorted heme configuration and a symmetric binding of the bulky nicotinate ligand.     

Ligand binding to heme proteins: II. Transitions in the heme pocket of myoglobin.

Phenomena occurring in the heme pocket after photolysis of carbonmonoxymyoglobin (MbCO) below about 100 K are investigated using temperature-derivative spectroscopy of the infrared absorption bands of CO. MbCO exists in three conformations (A substrates) that are distinguished by the stretch bands of the bound CO. We establish connections among the A substates and the substates of the photoproduct (B substates) using Fourier-transform infrared spectroscopy together with kinetic experiments on MbCO solution samples at different pH and on orthorhombic crystals. There is no one-to-one mapping between the A and B substates; in some cases, more than one B substate corresponds to a particular A substate. Rebinding is not simply a reversal of dissociation; transitions between B substates occur before rebinding. We measure the nonequilibrium populations of the B substates after photolysis below 25 K and determine the kinetics of B substate transitions leading to equilibrium. Transitions between B substates occur even at 4 K, whereas those between A substates have only been observed above about 160 K. The transitions between the B substates are nonexponential in time, providing evidence for a distribution of substates. The temperature dependence of the B substate transitions implies that they occur mainly by quantum-mechanical tunneling below 10 K. Taken together, the observations suggest that the transitions between the B substates within the same A substate reflect motions of the CO in the heme pocket and not conformational changes. Geminate rebinding of CO to Mb, monitored in the Soret band, depends on pH. Observation of geminate rebinding to the A substates in the infrared indicates that the pH dependence results from a population shift among the substates and not from a change of the rebinding to an individual A substate.     

Spectroscopic and interfacial properties of myoglobin/surfactant complexes

The complexes of horse myoglobin (Mb) with the anionic surfactant sodium dodecyl sulfate (SDS), and with the cationic surfactants cetyltrimethylammonium chloride (CTAC) and decyltrimethylammonium bromide (DeTAB), have been studied by a combination of surface tension measurements and optical spectroscopy, including heme absorption and aromatic amino acid fluorescence. SDS interacts in a monomeric form with Mb, which suggests the existence of a specific binding site for SDS, and induces the formation of a hexacoordinated Mb heme, possibly involving the distal histidine. Fluorescence spectra display an increase of tryptophan emission. Both effects point to an increased protein flexibility. SDS micelles induce both the appearance of two more heme species, one of which has the features of free heme, and protein unfolding. Mb/CTAC complexes display a very different behavior. CTAC monomers have no effect on the absorption spectra, and only a slight effect on the fluorescence spectra, whereas the formation of CTAC aggregates on the protein strongly affects both absorption and fluorescence. Mb/DeTAB complexes behave in a very similar way as Mb/CTAC complexes. The surface activity of the different Mb/surfactant complexes, as well as the interactions between the surfactants and Mb, are discussed on the basis of their structural properties.