Pentacoordinate Heme Research Articles

Formula omitted] NMR study of dynamics and thermodynamics of acid–alkaline transition in ferric hemoglobin of a midge larva ( Tokunagayusurika akamusi)

One of the components of hemoglobin from the larval hemolyph of Tokunagayusurika akamusi possesses naturally occurring substitution at the E7 helical position (Leu E7) [M. Fukuda, T. Takagi, K. Shikama, Biochim. Biophys. Acta 1157 (1993) 185–191]. Its oxygen affinity is almost comparable to those of mammalian myoglobins and it exhibits Bohr effect. Both acidic and alkaline forms of the ferric hemoglobin have been investigated using 1 H NMR in order to gain insight into molecular mechanisms for relatively high oxygen affinity and Bohr effect of this protein. The NMR data indicated that the acidic form of the protein possesses pentacoordinated heme, and that the alkaline form possessing OH − appears with increasing the pH value. pH titration yielded a p K value of 7.2 for the acid–alkaline transition, and this value is the lowest among the values reported so far for various myoglobins and hemoglobins. The kinetic measurements of the transition revealed that the activation energy for the dissociation of the Fe-bound OH −, as well as the dissociation and association rates, decrease with increasing the pH value. These pH dependence properties are likely to be related to the Bohr effect of this protein.

Charge reversal of a critical active-site residue of cytochrome-c peroxidase: characterization of the Arg48-->Glu variant.

A new variant of cytochrome-c peroxidase in which the positively charged Arg48 present in the distal heme-binding pocket has been replaced with a Glu residue has been prepared and characterized to explore, in part, the possibility that a negative charge close to the heme could contribute to stabilization of a porphyrin-centered pi-cation radical in the compound I derivative of the variant. Between pH 4 and 8, this variant forms three pH-linked spectroscopic species. The electronic absorption and 1H-NMR spectra of the predominant form at low pH (HS1) are indicative of a high-spin, pentacoordinate heme iron system. Near neutral pH, a second high-spin species (HS2) is dominant, in which the heme iron center is hexacoordinated, with a water molecule as the sixth axial ligand. At high pH, the third form (LS) exhibits the spectroscopic characteristics of a low-spin, hexacoordinate heme center with bishistidine axial ligation. The apparent pKa values for these transitions are 4.4 and 7.4, respectively, in phosphate buffers and 5.0 and 7.1, respectively, in phosphate/nitrate buffers. Replacement of Arg48 with Glu reduces the thermal stability of the enzyme and also decreases the Fe(III)/Fe(II) reduction potential of the enzyme by approximately 50 mV relative to that of the wild-type enzyme. The stability of compound I formed by the variant is decreased although the rate at which it forms is just one order of magnitude less than that of the wild-type enzyme, thus confirming previous results which indicate that the function of residue 48 in the wild-type peroxidase is more related to the stability of compound I than to its formation [Erman, J. E., Vitello, L. B., Miller, M. A. & Kraut, J. (1992) J. Am. Chem. Soc. 114, 6592-6593; Vitello, L. B., Erman, J. E., Miller, M. A., Wang, J. & Kraut, J. (1993) Biochemistry 32, 9807-9818]. Stopped-flow studies failed to detect even transient formation of a porphyrin-centered radical following addition of hydrogen peroxide to the Fe(III)-enzyme. The consequences of this drastic electrostatic modification of the active site on the steady-state kinetics of the variant are relatively minor.

Origin of the pH-dependent spectroscopic properties of pentacoordinate metmyoglobin variants.

The pH dependence of the electronic and EPR spectra of two variants of horse heart myoglobin (Mb) in which the distal His64 ligand has been replaced by either Thr or Ile has been studied. Both of these variants exhibit spectroscopic changes with pH that are indicative of a transition between two ferric high-spin forms that occurs with a pKa of 9.49 for the His64Thr variant and 9.26 for the His64Ile variant and that is distinctly different from the pH-dependent spectroscopic changes related to titration of the distal aquo ligand of wild-type Mb. The electronic and EPR spectra of both variants at all values of pH studied are consistent with the presence of a pentacoordinate heme iron center. For the His64Thr variant, a high-resolution (1.9 A) structure determination establishes the lack of the distal aquo ligand and demonstrates an out-of-plane movement of the ferric iron toward the proximal histidine together with a decrease of the Fe-His bond length. Investigation of this pH-linked equilibrium by EPR spectroscopy reveals rhombically split high-spin signals at both pH 7 and 11 with a greater degree of rhombicity exhibited by the alkaline species. We propose that the pH-linked spectroscopic transition exhibited by these distal histidine variants results from the deprotonation of the proximal His93 residue to produce imidazolate ligation at alkaline pH.

Structural characterization of oxidized dimeric Scapharca inaequivalvis hemoglobin by resonance Raman spectroscopy.

Resonance Raman spectra of the ferric homodimeric hemoglobin from Scapharca inaequivalvis have been measured over the pH range 5.8-8.3 in buffers of ionic strengths 0.01 and 0.1 M to determine the spin and coordination state of the iron atom. Three species contribute to the spectra: a low spin hexacoordinate, a high spin pentacoordinate, and a high spin hexacoordinate component. Optical absorption and EPR spectra measured under the same conditions allowed the identification of the ligands in the sixth coordination position, namely the distal histidine in the low spin derivative and a water molecule in the high spin one. The relative concentrations of these three species depend on pH in an unusual way. Thus, the aquomet derivative is present over the whole pH range, albeit in small amounts as most of the hemoglobin converts to the low spin hemichrome at acid pH values and to the pentacoordinate derivative at neutral and slightly alkaline ones. The formation of a pentacoordinate heme as the pH is increased has not been reported previously for other myoglobins and hemoglobins. Low ionic strength and high protein concentration favor the formation of the high spin pentacoordinate species, while at high ionic strength and low protein concentration the low spin hexacoordinate species prevails. Ionization of the iron-bound water molecule occurs at pH > or = 9.3; accordingly, signals from the hydroxyl derivative were not observed in the Raman spectra over the pH range studied.

Strong-field and integral spin-ligand complexes of the cytochrome bo quinol oxidase in Escherichia coli membrane preparations

The cytochrome bo-type terminal oxidase of Escherichia coli is an analogue of mammalian aa 3-type cytochrome c oxidase. The catalytic core of both enzymes is a binuclear site containing a penta-coordinate heme (heme o or a 3) and copper (Cu B). Herein we report on UV-visible and magnetic properties of ligand complexes of the binuclear site of cytochrome bo. Cyanide, sulfide, and azide react with the Fe 3+-Cu +center to give EPR-detectable low-spin complexes, analogous to those formed by cytochrome aa 3. Analyses of the ligand fields of these complexes indicate that heme o has a single axial histidine ligand. Cyanide and azide react with the Fe 3+ -Cu 2+ center to yield forms observable via UV-visible spectroscopy but not EPR. With formate and fluoride, cytochrome bo forms integral spin complexes similar to those of cytochrome aa 3. These complexes have UV-visible characteristics of high-spin species, but EPR spectra show features which appear to correspond to transitions within an integral spin multiplet. Cytochrome bo forms another integral spin complex with azide and NO which is nearly identical to the azide-NO species in cytochrome aa 3. This suggests that the binuclear centers of the two enzymes are quite similar.

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 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.

Dynamics and thermodynamics of acid-alkaline transitions in metmyoglobins lacking the usual distal histidine residue

The kinetics of the acid-alkaline transition in the ferric myoglobins from the gastropodic mollusc Dolabella auricularia and the shark Mustelus japonicus, which possess the distal Val E7 and Gln E7, respectively, has been investigated using the paramagnetic 1H-NMR saturation transfer measurements in order to gain insight into functional properties of these non-His distal residues. Both myoglobins possess the penta-coordinated heme below the p K of the transition (7.8 and 10.0 for Dolabella and Mustelus myoglobins, respectively) and bind OH − above the p K. The pH dependence of the transition rates and the relatively high activation barrier (58±9 kJ/mol) for the dissociation of the Fe-bound OH − in Dolabella myoglobin indicate a strong interaction between the bound ligand and the guanidino NH proton of the Arg E10 in Dolabella myoglobin. Such a strong interaction between Fe-bound OH − and the Arg E10 side-chain Dolabella myoglobin is also manifested in the EPR spectra. For Mustelus myoglobin, the pH and temperature dependence studies on the kinetics strongly suggest that the distal Gln E7 in this myoglobin does not contribute significantly to stabilize the Fe-bound ligand.

Horse heart ferricytochrome c: conformation and heme configuration of high ionic strength acidic forms.

The absorption, circular dichroism, and resonance Raman spectra of horse heart ferricytochrome c in the presence of 0.2 M KCl, 0.1 M NaClO4, and 0.2 M KNO3, in the pH region 7 to 0.5, have been investigated to determine the nature and the course of the processes involved. As in the absence of salts (Myer, Y., and Saturno, A. F. (1990) J. Protein Chem, 9, 379-387), the change from neutral to low acidic pH's in the presence of salts is a three-step process: state IIIs----state IIIS,a----state IIS----state IS, with pKa's of 3.5 +/- 0.2, 2.2 +/- 0.2, and 1.1 +/- 0.2, and with two, one, and one number of protons, respectively. The addition of salts at neutral pH's has little or no effect on the protein conformation and the heme-iron configuration (i.e., they remain the same, low-spin hexacoordinated heme iron with a Met-80-Fe-His-18 axial coordination), but such addition does cause a slight tightening of the heme crevice and the enlargement of the porphyrin core. State IIIS,a is a folded state with about the same degree of folding and with a similar spin state and coordination configuration of iron, but the heme crevice is loosened and the porphyrin core is smaller. Both states IIS and IS are also essentially folded forms, but with a smaller degree of protein secondary structure. State IIS has a high-spin hexacoordinated heme iron with a water molecular and a protonated and/or hydrogen-bonded imidazole of his-18 as the two axial ligates; and state IS has a high-spin pentacoordinated heme iron, which is about 0.49 A out of the porphyrin plane, with a protonated and/or hydrogen-bonded imidazole nitrogen as the only axial ligate. The addition of anions causes the stabilization of the protein secondary structures and the state IIIa----state II transition. The mode of effectiveness of anions appears to be nonspecific (i.e., because of electrostatic shielding and/or disruption of salt bridges).

Unusual carbon monoxide bonding geometry in abnormal subunits of hemoglobin M Boston and hemoglobin M Saskatoon

To clarify the role of the proximal histidine (F8-His), distal His (E7-His), and E11 valine (E11-Val) in ligand binding of hemoglobin (Hb), we have investigated the resonance Raman (RR) spectra of the carbon monoxide adduct of Hbs M (COHb M) in which one of these residues was genetically replaced by another amino acid in either the alpha or beta subunit. In the fully reduced state, all Hbs M gave v3 at approximately 1472 cm-1 and vFe-His at 214-218 cm-1, indicating that they have a pentacoordinate heme and the heme iron is bound to either E7-His or F8-His. The porphyrin skeletal vibrations of the COHb M were essentially unaltered by replacements of E7- or F8-His with tyrosine (Tyr) and of E11-Val by glutamic acid (Glu). The vCO, vFe-CO, and delta Fe-C-O frequencies of COHb M Iwate (alpha F8-His----Tyr), COHb M Hyde Park (beta F8-His----Tyr), and COHb M Milwaukee (beta E11-Val----Glu) were nearly identical with those of COHb A. In contrast, the RR spectra of COHb M Boston (alpha E7-His----Tyr) and COHb M Saskatoon (beta E7-His----Tyr) gave two new Raman bands derived from the abnormal subunits, vFe-CO at 490 cm-1 and vCO at 1972 cm-1, in addition to those from the normal subunits at 505 cm-1 (vFe-CO) and 1952 cm-1 (vCO). The CO adduct of the abnormal subunits exhibited apparently no photodissociation upon illumination of CW laser with a stationary cell under which the normal subunit exhibited complete photodissociation.(ABSTRACT TRUNCATED AT 250 WORDS)

NMR study of Galeorhinus japonicus myoglobin

The heme molecular structure of the met-azido form of the myoglobin from the shark Galeorhinus japonicus has been investigated by 1H NMR. A nuclear Overhauser effect (NOE) was clearly observed among the heme peripheral side-chain proton signals of this complex, which undergoes thermal spin equilibrium between high-spin (S = 5/2) and low-spin (S = 1/2) states, and the NOE connectivities provided the assignment of the resonances from the heme C13(1)H2 and C17(1)H2 protons. Chemical shift inequivalence of these proton resonances not only provided information about the orientation of these methylene protons with respect to the heme plane, but also allowed characterization of the time-dependent build-up of the NOE between them, which yields the correlation time for the internal motion of the inter-proton vector. The relatively large mobility found for the C17(1)H2 group suggests that the carboxyl oxygen of the heme C17 propionate is not anchored to the apo-protein by a salt bridge. It has been shown that the ferric high-spin form of G. japonicus Mb possesses a penta-coordinated heme [Suzuki, T. (1987) Biochim. Biophys. Acta 914, 170-176; Yamamoto, Y., Osawa, A., Inoue, Y., Chûjô, R. & Suzuki, T. (1990) Eur. J. Biochem. 192, 225-229] and that the conformation of both heme propionate groups is fixed with respect to the heme, as well as the apo-protein, by a salt bridge [Yamamoto, Y., Inoue, Y., Chûjô, R. & Suzuki, T. (1990) Eur. J. Biochem. 189, 567-573]. Therefore the weakening or interruption of the interaction between the C17 propionate and His FG3 upon the changes of the coordination and spin state of the heme iron, during azide ion binding to ferric high-spin G. japonicus Mb, is attributed to the displacement of the FG corner of the apoprotein away from the heme C17 propionate group. A similar structural alteration has been revealed by X-ray structural analyses of unliganded and liganded forms of ferrous hemoproteins [Baldwin, J. & Chothia, C. (1979) J. Mol. Biol. 129, 175-220; Phillips, S.E.V. (1980) J. Mol. Biol. 142, 531-554].

Horse heart ferricytochromec: Conformation and heme configuration of low ionic strength acidic forms

Resonance Raman, absorption and circular dichroism spectroscopic studies of the stable forms of horse heart ferricytochrome c in the pH range 6-0.8 and at the lowest possible ionic strengths, in water, and at 30 degrees C are reported. The neutral pH form, state III, changes to the acidic pH form, state I, through a three-step process: state III in equilibrium with IIIa in equilibrium with state II in equilibrium with state I, with pKa's of 3.6 +/- 0.3, 2.7 +/- 0.2, and 1.2 +/- 0.2, depending on the monitoring probe, respectively. State IIIa ferricytochrome c is like state III (i.e., with the Met-80-sulfur-iron linkage and a closed heme crevice) but with a higher degree of folding and a slightly larger porphyrin core. State II ferricytochrome c is an unfolded form with an open heme crevice and no Met-80-sulfur-iron linkage. The heme iron is high-spin, and hexacoordinated with weak ligand-field groups, water, and nitrogen of the protonated/hydrogen-bonded imidazole of the His-18 residue at the axial positions. The state I form also lacks the Met-80-sulfur-iron linkage and has an open heme crevice like the state II form; however, it is less unfolded and has a high-spin pentacoordinated heme iron, with the nitrogen of the imidazole of His-18 as the axial ligate, which is out of the porphyrin plane by about 0.45 A.

Resonance Raman studies of iron spin and axial coordination in distal pocket mutants of ferric myoglobin.

We have used resonance Raman spectroscopy to study 11 distal pocket mutants and the "wild type" and native ferric sperm whale myoglobin. The characteristic Raman core-size markers v4, v3, v2, and v10 are utilized to assign the spin and coordination state of each sample. It is demonstrated that replacements of the distal and proximal histidines can discriminate against H2O as a sixth ligand and favor a pentacoordinate Fe3+ atom. Soret absorption band blueshifts are correlated with the pentacoordinate heme environment. One E7 replacement (Arg) leads to an iron spin state change and produces a low spin species. The Glu and Ala mutations at position E11 leave the protein's spin and coordination unaltered. A laser-induced photoreduction effect is observed in all pentacoordinate mutants and seems to be correlated with the loss of the heme-bound water molecule.

A comparison of CO bound to metalloporphyrins and metal surfaces

A thorough understanding of the metalligand bond is of great importance in biochemistry as well as in catalysis. Much insight can be gained by an analysis of the various factors that govern the properties of this bond for a model system such as carbon monoxide bound to iron in heme or iron embedded in a metal surface. [1]. The delocalized sp-states in a metal show a large dispersion and form an electron gas whereas the Fe 4s electrons in heme reside in the surrounding porphyrin. The phorphyrin, basically a poly-pyrrol and thus ‘metallic’, can transfer long-range effects connecting substituents in the periphery with the FeCO or FeOO complex. Such properties can be simulated by a model in which iron is embedded in an electron gas but with different density for metalloporphyrins compared to metals. To what extent the response of such an electron gas to an external field will influence the matrix elements of photoexcitations is not known. The 3rd states of a typical transition metal such as iron are well localized within the Wigner-Seitz cell. It is thus plausible that e.g. a CO molecule is bound to a particular surface atom since the bond is believed to be combined CO5σFe3dσ and Fe3dπCO2 bond. The different crystal field separations of 3d states in the reactive pentacoordinated heme compared to the hexacoordinated hemeCO complexes is parallelled by the band-narrowing of 3d-states in the surface layer of a metal. Any change of the 3d states either in metalloporphyrins or in metal surfaces will influence the chemical properties since the changes occur close to the Fermi level, i.e. the mean energy of the highest occupied and lowest unoccupied molecular orbitals. The stretch frequencies of the metalcarbon ( v MC) and carbonoxygen ( v CO) bonds are sensitive probes of these changes. The analysis of such vibrational shifts in terms of electronic structure is however not always straightforward. A decreased redox potential (work-function) is followed by a decreased v CO [2, 3]. This can be interpreted in terms of an enhanced backbonding to the antibonding COπ level close to the Fermi level. The work-function decrease is provided by preadsorbed potassium on an iron catalyst which means that the we cannot completely rule out the possibility of direct COK interaction. In heme models, where the decrease is provided by substitutions in the periphery of the porphyrin, the pyrrols separate the substituents from the CO ligand and direct overlap is negligible. Moreover at high CO coverages of the metal surface we have the possibility of direct COCO overlap and formation of CO2π electron bands which will influence the occupation numbers and thus the vibrational frequencies [4, 5]. At intermediate coverages where ordered overlayers occur the v MC frequency can be influenced by phonon bands. The isolation of the direct FeCO electronic effects is however possible for a new model systems. One such system is CO bound to iron adsorbed at dilute coverages onto aluminium, a metal which is inert to CO. Another system is heme solvation as well as steric effects or H-bonds to a protein can be ruled out. A comparison of iron and copper porphyrins is valuable. In a FeCO complex the π states will be heavily mixed whereas in CuCO the effect will be merely a broadened 2π level. This is plausible since a copper surface or a copper porphyrin has a low density of states at the Fermi level compared to iron due to an almost completely filled 3d shell.

The Preparation of Protoheme Mono-N-[5-(2-methyl-1-imidazolyl)pentyl]amide and Its Oxygenation

Abstract Iron(II)protoporphyrin IX N-[5-(2-methyl-1-imidazolyl)pentyl]amide ethyl ester (1) and its derivatives (2–4) were prepared. They predominantly form pentacoordinate heme complexes in organic and aqueous solutions, based on their 2-methylimidazole-ligand and spacer N-pentylamide groups. Oxygen adducts of 1–4 were rapidly formed on exposure to oxygen in N,N-dimethylformamide (DMF) at −30 °C, and their life-times were more than 30 min.

Energy-structure correlation in metalloporphyrins and the control of oxygen binding by hemoglobin.

The contribution of the porphyrin skeleton to the potential energy surface metalloporphyrins is calculated by the semiempirical method of quantum mechanical extension of the consistent force field to eta electron molecules. This calculation makes it possible to correlate the observed structure of metalloporphyrins with the strain energy of the porphyrin skeleton. It is found that the out-of-plane metal displacement in pentacoordinate heme systems is due to both the restricted size of the porphyrin hole and the "1-3" steric interaction between the axial ligand and the heme nitrogens. The main components of the active site of hemoglobin are simulated by a histidine-heme-oxygen system. The energy surface of this system provides a quantitative explanation for the control of ligand binding by hemoglobin. It is shown that the heme acts as a diaphragm, designed to provide simultaneous binding to the histidine and the sixth ligand under the steric requirements of the 1-3 interactions. The dependence of the hemoglobin potential surface on the distance between the proximal histidine and the heme plane is evaluated for the R and T states, using the calculated heme potential and the observed energy of heme-heme interaction.

Carbon monoxide binding to pentacoordinate mercaptide-heme complexes: kinetic study on models for cytochrome P-450.

Mercaptide anions form exclusively penta-coordinate heme complexes [RS-heme] in polar and nonpolar solution over a wide range of mercaptide concentration. These complexes have a Soret peak at 408 nm and a formation constant of about 2.5 X 10(4) M(-1), and combine with CO to give a CO-cytochrome P-450 type spectrum. Kinetics of CO binding to mercaptide-heme complexes [RS-heme] have been studied by the flash photolysis method. Characteristic constants for this reaction suggest close similarities between [CH3-(CH2)3-S-heme] and cytochrome P-450. The reaction of alkoxide anion with heme has also been examined but no evidence was found for the existence of the [RO-heme-CO[ species.