Eur. J. Biochem. 210,337-341 (1992) zyxwvutsr 0 FEBS 1992 zyxwvutsrqpo The Soret magnetic circular dichroism of ferric high-spin myoglobins A probe for the distal histidine residue Ariki MATSUOKA, Nagao KOBAYASHI and Keiji SHIKAMA Biological Institute and Pharmaceutical Institute, Tohoku University, Sendai, Japan (Received July 2/August 28, 1992) zyxwvutsrqp - EJB 92 0935 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 zyxwvu Paramecium caudatum of particular interest for the evolution of these proteins from protozoa to higher animals. In studies of myoglobin and hemoglobin molecules, much attention has been directed to the possible roles of the distal (E7) histidine residue. It has been suggested to act as a gate (Karplus and McCammon, 1986) or a swinging door (Johnson et al., 1989) for ligand entry into the heme pocket, and to stabilize the bound dioxygen by hydrogen-bond formation (Wittenberg et al., 1965; Phillips and Schoenborn, 1981). Fur- thermore, to facilitate the effective movement of a catalytic proton from the solvent to the coordinated dioxygen via the imidazole ring, it participates in a proton-relay mechanism for the autoxidation of oxymyoglobin to its met form (Suga- wara and Shikama, 1980; Shikama, 1985,1988; Shikama and Matsuoka, 1986). However, it is not a simple task to determine unequivocally whether or not a myoglobin or a hemoglobin contains the usual distal histidine residue, even if the amino acid sequence is known. This is particularly true for the proteins isolated from lower organisms, since their globins show no notable degree of sequence similarity with mammalian myoglobins and hemoglobins. Another criteria is therefore needed to iden- tify the distal (E7) histidine residue in these proteins in solu- tion. Shikama and Matsuoka (1989) have examined a dozen myoglobins from various species for their spectrophotometric properties, and found that the proteins can be divided into two groups by a significant difference in the positioning of the Soret band. The oxymyoglobin peaks are in very close positions, whereas the acidic met forms have separate peak Correspondence zyxwvutsrqpon ta K. Shikama, Biological Institute, Faculty of Abbreviation. MCD, magnetic circular dichroism. Science, Tohoku University, Sendai, Japan 980 positions in two different ranges depending on the presence or absence of the distal histidine residue. The myoglobin from Aplysia kurodai shows a Soret peak that is profoundly blue shifted and accompanied by a marked intensity decrease, which may be due to a broadening of the spectrum. So far in all cases we have examined, this spectral feature is unique for myoglobins lacking the distal histidine residue, and a possible explanation seems to be as follows. In the acidic met- myoglobins, the sixth-(distal)-coordinate position of the ferric heme iron is usually occupied by a water molecule, which is hydrogen bonded to the distal histidine. In the case of Aplysia myoglobin, the distal position is vacant in its acidic met form, and the water molecule is found near Val63 at position E7, far from the heme iron (Giacometti et al., 1981 ; Bolognesi et al., 1989). This seems to explain the different spectral patterns, since the Soret band originates from a n -+ n* transition of the porphyrin ring and since this chromophore is likely to be open to more vibration, as well as to more doming toward the proximal side, due to lack of the axial-water ligand. In this respect, the magnetic circular dichroic (MCD) signal in the Soret region (350-450 nm) has been used as a more direct probe for the spin states and iron-coordination geometry in a wide variety of hemoproteins (Vickery et al., 1976; Holmquist, 1978; Bracete et al., 1991). In the present study, we evaluate the MCD spectra of ten native myoglobins and correlate the positions and intensities of their Soret peaks with the presence or absence of an axial water molecule, indicative of the distal (E7) histidine residue. On the basis of the MCD signals, we also examine a protozoan myoglobin (or monomeric hemoglobin) isolated from Para- mecium caudatum for its unique structure.