Sixth Coordination Position Research Articles

African elephant myoglobin with an unusual autoxidation behavior: comparison with the H64Q mutant of sperm whale myoglobin

Elephant myoglobins both from Asian and African species have a glutamine in place of the usual distal (E7) histidine at position 64. We have isolated native oxymyoglobin directly from the skeletal muscle of African elephant ( Loxodonta africana), and examined the autoxidation rate of oxymyoglobin (MbO 2) to metmyoglobin (metMb) as a function of pH in 0.1 M buffer at 25°C. As a result, African elephant MbO 2 was found to be equally resistant to autoxidation as sperm whale myoglobin. However, the elephant myoglobin exhibited a distinct rate saturation below pH 6. Kinetic analysis of the pH profiles for the autoxidation rate has disclosed that African elephant MbO 2 does not show any proton-catalyzed process, such as the one that can play a dominant role in the autoxidation reaction of sperm whale myoglobin by involving the distal histidine as its catalytic residue. Such a greater stability of African elephant MbO 2 at low pH could be explained almost completely by the single H64Q mutation of sperm whale myoglobin. In African elephant aqua-metmyoglobin the Soret band was considerably broadened so as to produce another peak in the pentacoordinate 395 nm region. This unique spectral feature was therefore analyzed to show that the myoglobin is in equilibrium between two species, depending upon the presence or absence of a water molecule at the sixth coordinate position.

Liposome effect on the cytochrome c-catalyzed peroxidation of carbonyl substrates to triplet species

Cytochrome c exhibits peroxidase activity on diphenylacetaldehyde (DPAA) and 3-methylacetoacetone (MAA), which is greatly affected by the presence and nature of charged liposome or micelle interfaces interacting with the enzyme. The ferricytochrome c reaction with DPAA is accelerated when the enzyme is attached to negatively charged interfaces. Whatever the medium, bulk solution or negatively charged dicetylphosphate (DCP), phosphatidylcoline/phosphatidylethanolamine/cardiolipin (PC/PE/CL) liposomes, this chemiluminescent reaction is accompanied by reduction of cytochrome c to its ferrous form. In turn, MAA is oxidized by cytochrome c exclusively when bound to DCP liposomes. Contrary to DPAA oxidation, the MAA reaction is followed by bleaching of cytochrome c, reflecting damage to the hemeprotein chromophore. The cytochrome- c-catalyzed oxidation of either DPAA or MAA leads to concomitant disappearance of the enzyme charge transfer absorption band at 695 nm. This suggests that the peroxidase activity of cytochrome c involves substrate-induced loss of the methionine ligand at the iron sixth coordination position, which is favored by interaction of cytochrome c with negatively charged interfaces. Accordingly, a decrease and blue shift of the charge transfer band could be observed in cytochrome- c-containing negatively charged DCP, PC/PE/CL liposomes or lysophosphatidylethanolamine micelles in the presence of DPAA or MAA.

Synthesis and structures of technetium(I) and rhenium(I) tricarbonyl complexes with bis(diphenylthiophosphoryl)amide, |M(CO) 3[(Ph 2PS) 2N](CH 3CN)| (M = Tc, Re)

Complexes of the general formula |M(CO) 3[(Ph 2PS) 2N](CH 3CN)| (M = Tc, Re) have been prepared by the reaction of (Et 4N) 2[M(CO) 3X 3] (M = Tc, Re; X = Cl, Br) with sodium bis(diphenylthiophosphoryl)amide, Na[(Ph 2PS) 2N], in acetonitrile. The metal is six-coordinate with facially arranged carbonyl ligands. Bis(diphenylthiophosphoryl)amide is deprotonated and bonded as S,S′-chelate with TcS distances of 2.526(3) and 2.546(3) Å. Acetonitrile occupies the sixth coordination position. The rhenium compound is isomorphous and the bonding situation in |Re(CO) 3[Ph 2PS) 2N](CH 3CN)| is similar to that in its technetium analogue. (Et 4N)[Re(CO) 3Br 2(CH 3CN)] crystallizes from a hot acetonitrile solution of (Et 4N) 2[Re(CO) 3Br 3] despite the fact that in solution only [Re(CO) 3(CH 3CN) 3] + can be detected spectroscopically. It has a facial arrangement of CO groups in an octahedral co-ordination geometry.

Covalent complex of microperoxidase with a 21-residue synthetic peptide as a maquette for low-molecular-mass redox proteins.

Here we report the structural and functional characterization of a covalent complex (MKP) obtained by cross-linking microperoxidase (Mp), the haem-undecapeptide obtained by the peptic digestion of cytochrome c, with a 21-residue synthetic peptide (P21) analogous to the S-peptide of the RNase A. The covalent complex has been prepared by introducing a disulphide bond between Cys-1 of P21 and Lys-13 of Mp, previously modified with a thiol-containing reagent. On formation of the complex (which is a monomer), the helical content of P21 increases significantly. The results obtained indicate that His-13 of P21 co-ordinates to the sixth co-ordination position of the haem iron, thus leading to the formation of a complex characterized by an equilibrium between an 'open' and a 'closed' structure, as confirmed by molecular dynamics simulations. Under acidic pH conditions, where His-13 of P21 is loosely bound to the haem iron ('open' conformation), MKP displays appreciable, quasi-reversible electrochemical activity; in contrast, at neutral pH ('closed' conformation) electrochemical behaviour is negligible, indicating that P21 interferes with the electron-transfer properties typical of Mp. On the whole, MKP is a suitable starting material for building a miniature haem system, with interesting potential for application to biosensor technology.

Synthesis and characterization of a dimeric complex of Cu II with thiosalicylic acid and pyridine

The interaction between the Cu 2+ cation and thiosalicylic acid was investigated. The solid complex is formed in methanolic solution and contains two Cu II ions bridged by carboxylate groups of four ligand molecules and pyridine ligands that occupy the sixth coordination positions. Spectral measurements (IR, Raman and electronic spectra) show the lack of interaction of the −SH group in coordination. These results are supported by elemental and thermogravimetric analysis as well as magnetochemical measurements.

Spectroscopic studies of the met form of the extracellular hemoglobin from Glossoscolex paulistus

Sephadex G-200 chromatography of the extracellular hemoglobin from the giant earthworm G. paulistus in the met form presents a single peak at pH 7.0 and two peaks at pH 9.0 as a result of alkaline dissociation. SDS-PAGE shows that the polypeptide chains are very similar to those observed for the oxy form and the two peaks at pH 9.0 correspond to the trimer contaminated by linkers and monomers which seems to be quite pure. The aquomet acid form is stable as an oligomer of molecular mass 3.1 × 10 6 Da only in a narrow pH range around neutrality. Increasing the pH above 7.5 leads to an irreversible transition from aquomet to hemichrome I which is the low-spin bis-imidazole complex. At pHs above 9.5–10.0 a second reversible transition takes place from hemichrome I to hemichrome II, a high-spin complex which is associated with the weakening and possible disruption of the proximal FeN histidine bond. Thus, increase in pH above 8.0 induces changes in the heme pocket that involve both the distal and proximal sides of the heme. EPR measurements show a very sharp decrease of the aquomet high-spin signal in the range of pH 7.0–8.0 and a very small low-spin signal even at liquid helium temperatures. The transition to hemichrome I is also accompanied by the loss of heme optical activity monitored by CD, which is consistent with the weakening of heme-globin interaction. Hemichrome I in the presence of cyanide gives the typical cyanometHb derivative which has a transition to a hemichrome at much higher pHs. This observation suggests that the dissociation of the oligomer in alkaline medium as well as the stability of the heme on the proximal side, depend both upon the ligand present at the sixth coordination position on the distal side. Hence, we believe that hemi(hemo)chrome formation in G. paulistus Hb and other invertebrate hemoglobins is a common phenomenon, not associated with protein denaturation, which may provide a fine tuning mechanism to control subunit interactions through changes in the distal side of the heme pocket.

Influence of KF, DCMU and removal of Ca + on the high-spin EPR signal of the cytochrome b-559 heme Fe(III) ligated by OH − in chloroplasts

An EPR signal at g = 6.8 attributed to the cytochrome (Cyt) b-559 heme Fe(III) ligated by OH − (Fiege, R., Schrieber, U., Lubitz, W., Renger, G. and Shuvalov, V.A. (1995) FEBS Lett. 377, 325–329) was studied. This signal is observed in intact chloroplasts when oxidized by 10 mM 2,3-dicyano,5,6-dichloro- p-benzoquinone (DDQ), but not when 5 mM p-benzoquinone is added. Addition of KF (100 mM) or removal of Ca 21 for blocking the water-oxidizing complex considerably decreases the heme Fe(III)-OH − EPR signal. In contrast, DCMU does not decrease this signal and does not influence its photochemical changes at 140 K. Thus, the EPR spectrum of Cyt b-559 Fe(III) ligated by OH − is not affected by changes at the acceptor side of Photosystem 11, and its photochemical decrease is probably not due to reduction via the acceptor side. Comparison of the effect of KF on the model heme Fe(III) in myoglobin (Mb) at pH 10.5 shows that F − replaces OH − as a ligand at the sixth coordination position of the heme Fe(III) in both Mb and chloroplasts Cyt b-559. This replacement is accompanied by changes of the symmetry of the molecular field causing a disappearance of the EPR signals at g = 6.8 and 5.0. Our results provide further evidence for a possible participation of the Cyt b-559 heme Fe ligated by OH − in photosynthetic water oxidation.

Mechanism of hydrogen cyanide binding to myoglobin.

Cyanide binding to myoglobin is much slower than that of other ferric and ferrous ligands, suggesting rate limitation by bond formation and disruption within the distal pocket. This interpretation is supported by two key experimental observations. First, His64(E7) to Gly and Ala mutations, which open a direct channel from the solvent to the iron atom, and Phe46(CD4) to Leu, Ile, and Val mutations, which increase the mobility of the distal histidine, have little effect on the association rate constant for cyanide binding. In contrast, these mutations cause 100-1000-fold increases in the rate constant for azide binding, showing convincingly that the binding of this ligand is limited by the rate of its movement into the protein. Second, the rate constant for cyanide dissociation is unaffected by changing the size of the residue at position 64(E7) in the series Gly, Val, Leu, Ile, Phe, whereas there is a 2000-fold decrease in the rate of azide dissociation in going from Gly64 to Phe64 metmyoglobin. The major determinants of the cyanide affinity are the ease of water displacement from the ferric iron atom in metmyoglobin, the acid dissociation constant of HCN inside the protein (K*a), and steric hindrance and electrostatic interactions at the sixth coordination position. Direct hydrogen bonding to the distal histidine does not appear to play an important role in stabilizing bound cyanide. Instead, the general polarity of the distal pocket and its effect on K*a are the key factors regulating cyanide affinity under physiological conditions.

NMR Studies of Substrate Binding to Cytochrome P450 BM3: Comparisons to Cytochrome P450 cam

The binding of the substrates sodium laurate and sodium 12-bromolaurate to the heme-containing domain of Bacillus megaterium cytochrome P450 BM3 (CYP102) has been studied by measurement of the relaxation effects of the unpaired electrons of the heme iron on the protons of water and of the bound substrates. Substrate binding leads to a conversion of the heme iron from a low-spin to a high-spin state, as shown by changes in the optical spectrum. The relaxation measurements show that this is accompanied by expulsion of water from the sixth coordination position of the iron, the distance between the iron and the water protons increasing from 2.6 to 5.2 A. Corresponding relaxation measurements on the substrate protons lead to the determination of a number of distances between the iron and protons of the bound substrate and, hence, to information on the position and orientation of the substrate in the binding site. Laurate and 12-bromolaurate are found to bind in a very similar way, in an extended conformation with the carboxylate probably close to Arg47 and the other end of the chain 7.6-7.8 A from the heme iron. It is shown that laurate and pyridine can bind simultaneously to the P450 domain and that the iron-laurate distances in this ternary complex are not significantly different from those in the binary complex. These observations are compared with those on the substrate complex of cytochrome P450 cam, and their implications for structural changes involved in the catalytic cycle are discussed.

A complex of microperoxidase with a synthetic peptide: structural and functional characterization

This paper reports the kinetic and thermodynamic characterization of the complex obtained by binding to microperoxidase (the heme-containing undecapeptide derived from peptic hydrolysis of cytochrome c) a 13 residues synthetic peptide with some propensity to acquire α-helical secondary structure (P13). Our results indicate that P13 binds to the sixth coordination position of the Fe(III) of microperoxidase ( K eq = 4.8 · 10 4M −1 at pH 7.0 and 25° C) via the imidazole of His-12, forming a stable complex. The kinetics of complex formation, its secondary structure and its electrochemical activity are reported. This is a first step towards engineering a miniature-heme complex, for a better understanding of the mechanisms governing electron transfer in hemes and heme-proteins, and possibly for novel biotechnological applications.

Spectral Characterization and Chemical Modification of Catalase-Peroxidase from Streptomyces sp.

Catalase-peroxidase was purified to near homogeneity from Streptomyces sp. The enzyme was composed of two subunits with a molecular mass of 78 kDa and contained 1.05 mol of protoporphyrin IX/mol of dimeric protein. The absorption and resonance Raman spectra of the native and its cyano-enzyme were closely similar to those of other heme proteins with a histidine as the fifth ligand. However, the peak from tyrosine ring at approximately 1612 cm-1, which is unique in catalases, was not found in resonance Raman spectra of catalase-peroxidase. The electron paramagnetic resonance spectrum of the native enzyme revealed uniquely two sets of rhombic signals, which were converted to a single high spin, hexacoordinate species after the addition of sodium formate. Cyanide bound to the sixth coordination position of the heme iron, thereby converting the enzyme to a low spin, hexacoordinate species. The time-dependent inactivation of the enzyme with diethyl pyrocarbonate and its kinetic analysis strongly suggested the occurrence of histidine residue. From the above-mentioned spectroscopic results and chemical modification, it was deduced that the native enzyme is predominantly in the high spin, ferric form and has a histidine as the fifth ligand.

Iron(II) complexes of polydentate aminopyridyl ligands and an exchangeable sixth ligand; reactions with peroxides. Crystal structure of [FeL1(H2O)][PF6]2·H2O [L1=N,N′-bis-(6-methyl-2-pyridylmethyl)-N,N′-bis(2-pyridylmethyl)ethane-1,2-diamine

Iron(II) complexes of the pentadentate ligand N-methyl-N,N′,N′-tris(2-pyridylmethyl)ethane-1,2-diamine (L2) and the potentially hexadentate ligand N,N′-bis(6-methyl-2-pyridylmethyl)-N,N′-bis(2-pyridylmethyl)-N,N′-bis(2-pyridylmethyl)ethane-1,2-diamine (L1) have been isolated. Both ligands behave as pentadentate ligands in their iron(II) complexes with the general formulation [FeL(X)]n+(L = L1 or L2, n= 1 or 2) with the sixth co-ordination site (X) occupied by an auxiliary ligand e.g. H2O, Cl, SCN or CN. The crystal structure of [FeL1(H2O)][PF6]2·H2O 1 shows that the nitrogen atom of one of the 6-methyl-2-pyridylmethyl groups of the formally hexadentate ligand is not co-ordinated to the iron atom. Instead a water molecule occupies the sixth co-ordination position. Complex 1 crystallizes in the triclinic space group P, with a= 10.485(2), b= 13.161(5), c= 14.853(6)A, α= 69.31(4), β= 74.42(3), γ= 66.37(3)° and Z= 2. The structure refined to a final R value of 0.052 for 2531 reflections. The complexes show peroxidase activity, the availability of a labile site on the relatively stable iron(II) complexes, along with no apparent tendency to form oxo-bridged diiron(III) species, which are likely to be crucial factors in the mechanism of the oxidation reactions. Efforts to isolate a complex with a co-ordinated peroxide in the sixth position were unsuccessful. However, we have spectroscopic evidence that peroxide reacts with the iron(II) complexes to form an unstable species, probably of the form [FeIIIL(OOR)]2+(L = L1 or L2, R = H or But). The peroxide ligand in this iron(III) complex is likely to be forced into an ‘end-on’ co-ordination mode.

Structure of the Sulfide-reactive Hemoglobin from the Clam Lucina pectinata: Crystallographic Analysis at 1.5 Å Resolution

The crystal structure of the aquo-met form of the sulfide-reactive hemoglobin (component I) from the gill of the symbiont-harboring mollusc, Lucina pectinata , has been and refined at 1.5 Å resolution, based on synchrotron X-ray diffraction data, and employing molecular replacement techniques. The crystallographic R-factor, calculated for the data in the 15.0 to 1.5 Å resolution range is 0.170 with highly regular stereochemical parameters for the protein model, and including 131 water molecules. The monomeric hemoglobin I chain consists of 142 amino acid residues, which have been partly identified on the basis of the crystallographic analysis. The molecule is characterized by an unusual distribution of aromatic residues, particular in the region surrounding the distal site in the heme pocket. The heme distal residue is Gln(64)E7, while other notable amino acid substitutions include Trp(21)B2, Phe(29)B10, Leu(46)CD3, Phe(68)E11 and Trp(75)E18. An amino acid insertion (Ser44) is observed between sites CD1 and CD2. In the aquo-met protein, a water molecule is present at the sixth coordination position of the heme iron, and hydrogen bonded to Gln(64)E7. Simple model building shows that dioxygen molecule, bound to ferrous protein, would contact with its free atom the ring edge of Phe(29)B10, being thus stabilized at the coordination site by an aromatic-electrostatic interaction. Similarly, the unique packing and organization of aromatic residues in the surroundings of the heme distal site is proposed as the molecular basis of the very high affinity of Lucina pectinata hemoglobin I for hydrogen sulfide, considered as one of the two physiological ligands of the protein.

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.

Comparison of heme environment at the putative distal region of P-450 s utilizing their external and internal nitrogenous ligand bound forms

Thr-303 to Lys-mutated P-450 2E1 1 1 Individual forms of P-450 are designated according to the nomenclature recommended by Nebert et al. [1]. , as well as Thr-301 to Lys-mutated P-450 2C2, had absorption spectra characteristic of a nitrogenous ligand-bound form of P-450, such as the pyridine complex of P-450 2E1; (i) in the ferric state, the red-shifted Soret band, compared with the typical low-spin type spectmm of P-450, and the more intense β band than the α band and (ii) in the ferrous state, two Soret peaks at around 447 and 422 nm, the relative intensities of which depended on pH, indicating the existence of two interconvertible states. The equilibrium between the two states of the mutated P-450 2E1 appeared to be shifted toward the 422 nm state, compared with the mutated P-450 2C2. The corresponding mutant of P-450 2C14 had similar spectral properties to those of both mutated P-450 s except that the shorter of the two Soret bands of its ferrous form was relatively broad and appeared at 418 mn. These findings suggest that the ϵ-amino-nitrogen of the Lys of the mutated P-450 s is located in the appropriate position to occupy the sixth coordination position with the heme iron and spatial differences exist in the essentially conserved structure of the distal heme domain among the three ferrous Lys-mutated P-450 s.

29] Measuring relative rates of hemoglobin oxidation and denaturation

Publisher Summary In aqueous media, without the active methemoglobin reductase system present in intact red blood cells, the ferrous heme groups of hemoglobin can auto-oxidize to form continually increasing amounts of high-spin ferric heme. Under conditions that promote tetramer dissociation, subsequent tertiary structural distortions in the globin monomers occur; these distortions are associated with iron spin-state transitions and possible geometric distortions of the heme moiety within individual heme pockets. Binding of low-spin ferric heme at the sixth coordinate position of the heme iron to normally distant amino acid side chains within the heme pocket can then result in the formation and accumulation of soluble and insoluble hemichromes. This chapter presents a generalized outline of this pathway. An alternate degradative path is also shown, wherein the high-spin ferric heme of hemoglobin is transferred to human serum albumin to form methemalbumin. The apoprotein thus formed is insoluble.

High-Resolution Crystal Structures of Distal Histidine Mutants of Sperm Whale Myoglobin

The highly conserved distal histidine residue (His64) of sperm whale myoglobin modulates the affinity of ligands. In an effort to fully characterize the effects of mutating residue 64, we have determined the high-resolution crystal structures of the Gly64, Val64, Leu64, Thr64 and Gln64 mutants in several liganded forms. Metmyoglobins with hydrophobic substitutions at residue 64 (Val64 and Leu64) lack a water molecule at the sixth coordination position, while those with polar amine acid residues at this position (wild-type and Gln64) retain a covalently bound water molecule. In the Thr64 mutant, the bound water position is only partially occupied. In contrast, mutating the distal histidine residue to glycine does not cause loss of the coordinated water molecule, because the hydrogen bond from the imidazole side-chain is replaced by one from a well-ordered solvent water molecule. Differences in water structure around the distal pocket are apparent also in the structures of deoxymyoglobin mutants. The water molecule that is hydrogen-bonded to the Nε atom of histidine 64 in wild-type deoxymyoglobin is not found in any of the position 64 mutant structures that were determined. Comparison of the carbonmonoxy structures of wild-type, Gly64, Leu64 and Gln64 myoglobins in the P 6 crystal form shows that the conformation of the Fe-C-O complex is nearly linear and is independent of the identity of the amino acid residue at position 64. However, the effect of CO binding on the conformation of residue 64 is striking. Superposition of deoxy and carbonmonoxy structures reveals significant displacements of the residue 64 side-chain in the wild-type and Gln64 myoglobins, but no displacement in the Leu64 mutant. These detailed structural studies provide key insights into the mechanisms of ligand binding and discrimination in myoglobin.

Crystal structure of a distal site double mutant of sperm whale myoglobin at 1.6 Å resolution

The three-dimensional structure of sperm whale myoglobin His 64(E7)→Val,Thr 64(E10)→Arg double mutant has been studied by X-ray crystallography at 1.6 Å resolution, and refined to a crystallographic R-factor of 0.197. The Arg 67(E10) side chain is extended in the direction of the ligand binding site, and its NH1 atom is at a distance of 3.11 Å from the NH1 atom of Arg 45(CD3), which is also pointing towards the distal site. Both are kept in this position by hydrogen bonding and electrostatic interactions with a solvent sulfate ion, located amongst the two, on the protein surface. No liganded water molecule is present at the sixth coordination position of the Fe(III) heme.

Haem–peptide complexes. Synthesis and stereoselective oxidations by deuterohaemin-L-phenylalanyl-poly-L-alanine complexes

Deuterohaemin-L-histidine methyl ester—peptide complexes have been obtained by covalently linking L-histidine methyl ester and the peptides Ala-Ala-Phe-Ala-Ala-Ala-Ala-Ala-Ala-Ala (compound 3) or Ala-Ala-Ala-Phe-Ala-Ala-Ala-Ala-Ala-Ala (compound 4) on the propionic acid side chains. The spectroscopic properties of 3 and 4 and the imidazole binding equilibria indicate that folding of the peptide chain on the opposite part of the porphyrin plane with respect to that occupied by the bound histidine side-arm reduces in the order 3 > 4 the accessibility of exogenous ligands to the sixth co-ordination position of the iron atom, probably through some stacking interactions between the phenylalanine residue of the peptide and the porphyrin ring in the case of 3. This arrangement has marked consequences on the stereoselectivity observed in a model peroxidase reaction using L- or D-tyrosine methyl ester as substrates and tert-butyl hydroperoxide as oxidant in dichloromethane–trifluoroethanol (9:1), that have been interpreted in terms of the interaction between the chiral substrates and the peptide chains of the deuterohaemin complexes.

The Soret magnetic circular dichroism of ferric high-spin myoglobins. A probe for the distal histidine residue.

To find a simple criterion for the presence of the distal (E7) histidine residue in myoglobins and hemoglobins, the Soret magnetic-circular-dichroic spectra were examined for ferric metmyoglobins from various species. A distinct and symmetric dispersion-type curve was obtained for myoglobins containing the distal histidine, whereas a relatively weak and unsymmetric pattern was observed for myoglobins lacking this residue, such as those from three kinds of gastropodic sea molluscs, a shark and the African elephant. The magnetic-circular-dichroic spectra obtained would thus be a direct reflection of the presence or absence of a water molecule at the sixth coordinate position of the heme iron(III), this axial water ligand being stabilized by hydrogen-bond formation to the distal histidine residue. On the basis of these Soret magnetic-circular-dichroic signals, we also examined the structure of a protozoan myoglobin (or a monomeric hemoglobin) from Paramecium caudatum of particular interest for the evolution of these proteins from protozoa to higher animals.