Oxidation of Residue 45 Mutant Forms of Pig Deoxymyoglobin With [Fe(CN),13- zyxwvutsrqponmlkjih BaoJian Zhang, Stephen J. Smerdon,* Anthony J. Wilkinson,* and A. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Geoffrey Sykes Department of Chemist-y, The Universiiy, Newcastle upon Tyne, U.K. ABSTRACT The effect of replacement of the highly conserved Lys45 residue in pig myoglohin (Mb) with His, Ser, Glu, and Arg has been investigated. Rate constants/M-‘s-’ at 25°C and pH 8.0, I = 0.100 M (NaCI), for the oxidation of deoxyMh with [Fe(CN),13- have been determined, and are for wild-type Lys45 (2.83 x 106), His45 (1.02 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIH x lo’?, Ser45 (1.12 X 106), Glu45 (0.87 X 106), and Arg45 (3.06 x 106). It is concluded that charge on the residue at position 45 has only a mild effect on reactivity, and that this is unlikely to he the site for electron transfer. INTRODUCTION Virtually all mammalian myoglobins have a lysine residue at position 45 (CD3); exceptions are sperm whale and aardvark myoglobins which have arginine at this position [l]. Molecular dynamic calculations have suggested that. ligands which bind to the Fe enter and exit the heme pocket through a pathway between Va168 (El11 and the distal histidine His64 (E7) (Fig. 1). 12, 31. The Lys45 residue lies between His64 and external solvent [41, and it has been postulated that electro- static and polar interactions involving these groups and surface water molecules might serve as a barrier to ligand entry into the heme pocket [S]. However, mutations at position 45 do not change significantly (< 2-fold) the overall association rate constants for NO, CO, and 0, binding at room temperature [l]. A 3-fold decrease in the initial rate for intramolecular picosecond rebinding of NO, and a 4-fold decrease in the geminate rate constant for the nanosecond rebinding of 0, have been observed 111. The 0, affinity of the Glu45 mutant is 4-fold lower than that of the Arg45 mutant due primarily to an increase in the * Department of Chemistry, The University, York, YO15DD, U.K. Address reprint requests to: Professor A. G. Sykes, Department of Chemistry, Bedson Building, The University, Newcastle upon ‘Qne, NE1 7RU, U.K. Journal zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA of Inorganic Biochemkhy,48,79-&I (1992) 19 8 1992 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, NY, NY 10010 0162-0134/92/.$5.00