Pronounced Conversion of the Metal-Specific Activity of Superoxide Dismutase from Porphyromonas gingiValis by the Mutation of a Single Amino Acid (Gly155Thr) Located Apart from the Active Site †,‡ Fumiyuki Yamakura,* ,§,| Shigetoshi Sugio, |,⊥ B. Yukihiro Hiraoka, |,@ Daijiro Ohmori, § and Takehiro Yokota ⊥ Department of Chemistry, Juntendo UniVersity School of Medicine, Inba 270-1695, Japan, Science & Technology Research Center, Mitsubishi Chemical Corporation, 1000 Kamoshida, Aoba, Yokohama 227-8502, Japan, and Institute for Oral Science, Matsumoto Dental UniVersity, Shiojiri 399-0781, Japan ReceiVed June 4, 2003; ReVised Manuscript ReceiVed July 30, 2003 ABSTRACT: Glycine 155, which is located ∼10 Å from the active metal sites, is mostly conserved in aligned amino acid sequences of manganese-specific superoxide dismutases (Mn-SODs) and cambialistic SOD (showing the same activity with Fe and Mn) from Porphyromonas gingiValis, but is substituted for threonine in most Fe-SODs. Since Thr155 is located between Trp123 and Trp125, and Trp123 is one member of the metal-surrounding aromatic amino acids, there is a possibility that the conversion of this amino acid may cause a conversion of the metal-specific activity of cambialistic P. gingiValis SOD. To clarify this possibility, we have prepared a mutant of the P. gingiValis SOD with conversion of Gly155 to Thr. The ratios of the specific activities of Fe- to Mn-reconstituted enzyme, which are measured by the xanthine oxidase/cytochrome c method, increased from 0.6 in the wild-type to 11.2 in the mutant SODs, indicating the conversion of the metal-specific activity of the enzyme from a cambialistic type to an Fe-specific type. The visible absorption spectra of the Fe- and Mn-reconstituted mutant SODs closely resembled those of Fe-specific SOD. Furthermore, the EPR spectra of the Fe- and Mn-reconstituted mutant SODs also closely resembled those of Fe-specific SOD. Three-dimensional structures of the Fe-reconstituted wild-type SOD and Mn-reconstituted mutant SOD have been determined at 1.6 Å resolution. Both structures have identical conformations, orientations of residues involved in metal binding, and hydrogen bond networks, while the side chain of Trp123 is moved further toward the metal-binding site than in wild- type SOD. A possible contribution of the structural differences to the conversion of the metal-specific activity through rearrangement of the hydrogen bond network among Trp123, Gln70, Tyr35, and the metal-coordinated solvent is discussed. Four main classes of superoxide dismutase (SOD) 1 have been reported, each differing in their metallic cofactors: copper- and zinc-containing (Cu,Zn-SOD), iron-containing (Fe-SOD), manganese-containing (Mn-SOD), and nickel- containing (Ni-SOD) SODs. Among these SODs, Fe-SOD and Mn-SOD can be divided into two types based on metal specificity for the enzymatic reaction. The first is a metal- specific type of SOD, which requires the original metals for the activity; that is, manganese-substituted Fe-SODs (1) and iron-substituted Mn-SODs (2, 3) retain little or no enzymatic activity. Fe-SOD from Pseudomonas oValis (1) and Fe- and Mn-SOD from Escherichia coli (2, 3) are typical examples of this type of SOD. The other type of SOD, cambialistic SOD (4-7), uses both metals to exhibit the enzymatic activity. Despite these differences in metal specificity, Mn-SODs (8-10), Fe-SODs (11, 12), and cambialistic SODs (13, 14) have a large degree of sequence homology and X-ray structural similarity. The iron and manganese atoms are commonly ligated by three histidine residues, one aspartate residue, and one solvent molecule. Since both Fe and Mn can mediate dismutation reactions of superoxide, and since specificity of metals can be different according to the type of SODs, there must be some subtle but critical differences in the interaction between the proteins and the metal ion. Therefore, Fe- and Mn-SODs provide an ideal model for analyzing how a metalloenzyme precisely tunes its metal site suitably for metal-substrate interaction. Although no significant difference has been observed at the active site of these SODs, two minor differences were observed in the second sphere of the active site (within 8 Å) of the enzymes. One difference is that Gln70 in Fe-SODs is complementarily substituted with Gln142 in Mn-SODs (note that the amino acid numbering is based on the positions in Porphyromonas gingiValis SOD), with the same orienta- † This work was partially supported by the National Project on Protein Structural and Functional Analyses run by the Japanese Ministry of Education, Culture, Sports, Science and Technology. ‡ PDB entries 1UER for the wild-type Fe-SOD and 1UES for the mutant Mn-SOD. * To whom correspondence should be addressed. Telephone: +81- 476-98-1001. Fax: +81-476-98-1011. E-mail: yamakura@sakura. juntendo.ac.jp. § Juntendo University School of Medicine. | These authors contributed equally to this work. ⊥ Mitsubishi Chemical Corp. @ Matsumoto Dental University. 1 Abbreviations: SOD, superoxide dismutase; Fe-SOD, iron-contain- ing superoxide dismutase; Mn-SOD, manganese-containing superoxide dismutase; MBP, maltose binding protein. 10790 Biochemistry 2003, 42, 10790-10799 10.1021/bi0349625 CCC: $25.00 © 2003 American Chemical Society Published on Web 08/21/2003