Three residues His29, His84 and His171 were found to be the ligands of metal ions of one hyperthermostable Mn-SOD produced by Thermus thermophilus HB27 after multiple sequence alignment. Conversion of these residues might cause a change of metal-binding specificity. Thus, three mutants His29Ala, His84Ala, and His171Ala were prepared. The mutant types changed, and the metal-binding specificities of His29Ala and His171Ala altered from Mn to Fe. The alpha-helix contents of His29Ala and His171Ala were 66% and 59% respectively, suggesting that the mutants folded with a reasonable secondary structure. His29Ala showed an activity comparable to that of the wild type, and His171Ala exhibited a 39.6% higher activity than the wild type. These two mutants were thermostable from 30 to 50 degrees C after incubation for 30 min. The activity of His171Ala retained 51% at 70 degrees C compared with 36.7% for His29Ala. Both mutants retained >80% residual activity in the pH range 5.0-10.0 (except their pI of 8.0) after incubation at 4 degrees C for 30min. This study provides a good foundation for accelerating the development of thermostable Fe-SOD for application in the fields of medicine and cosmetics. (C) 2015 Elsevier Ltd. All rights reserved.