Time courses for the autooxidation of native and mutant sperm whale and pig myoglobins were measured at 37$\sp\circ$C in the presence of catalase and superoxide dismutase. In sperm whale myoglobin, His$\sp{64}$ (E7) was replaced with Gln, Gly, Val, Leu, and Phe; Val$\sp{68}$ (E11) was replaced with Ala, Ile, and Phe; Leu$\sp{29}$ (B10) was replaced with Ala, Val, and Phe. In pig myoglobin, His$\sp{64}$ (E7) was replaced with Val; Val$\sp{68}$ (E11) was replaced with Thr and Ser; Thr$\sp{67}$ (E10) was replaced with Ala, Val, Glu, and Arg; Lys$\sp{45}$ (CD3) was replaced with Ser, Glu, His, and Arg. The observed pseudo first order rate constants varied 5 orders of magnitude, from 44 hr$\sp{-1}$ (H64G) to 0.055 hr$\sp{-1}$ (native) to 0.005 hr$\sp{-1}$ (L29F) at 37$\sp\circ$C, pH 7 in air. The dependence of autooxidation on oxygen and pH were measured for selected proteins.
In the native proteins and in most mutants still possessing the distal histidine, autooxidation occurs through a combination of two mechanisms. At high oxygen levels, autooxidation proceeds by direct dissociation of the neutral superoxide radical (HO$\sb2$) from oxymyoglobin, and this process is accelerated by decreasing pH. At low oxygen levels, autooxidation also occurs by a bimolecular reaction between molecular oxygen and a very weakly bound complex between water and ferrous deoxymyoglobin. The neutral side chain of the distal histidine (His$\sp{64}$) inhibits autooxidation by hydrogen bonding to bound oxygen. Replacement of His$\sp{64}$ by amino acids incapable of hydrogen bonding to the bound ligand causes a change in the mechanism of autooxidation and marked increases in the rate of autooxidation. Increasing the polarity of the distal pocket by substitution of Val$\sp{68}$ with Ser and Thr accelerates autooxidation, presumably by facilitating protonation of the Fe(II)-O$\sb2$ complex. Increasing the net anionic charge at the protein surface in the vicinity of the heme group also enhances the rate of autooxidation. Decreasing the volume of the distal pocket by replacing small amino acids with larger aliphatic or aromatic residues at positions 68 (E11) and 29 (B10) inhibits autooxidation markedly by decreasing the accessibility of the iron atom to solvent water molecules.
| Identifer | oai:union.ndltd.org:RICE/oai:scholarship.rice.edu:1911/16602 |
| Date | January 1992 |
| Creators | Brantley, Robert Earl, Jr |
| Contributors | Olson, John S. |
| Source Sets | Rice University |
| Language | English |
| Detected Language | English |
| Type | Thesis, Text |
| Format | 131 p., application/pdf |
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