Role of disulfide bonds in the stability of recombinant manganese peroxidase.
Phanerochaete chrysosporium manganese peroxidase (MnP) [isoenzyme H4] was engineered with additional disulfide bonds to provide structural reinforcement to the proximal and distal calcium-binding sites. This rational protein engineering investigated the effects of multiple disulfide bonds on the stabilization of the enzyme heme environment and oxidase activity. Stabilization of the heme environment was monitored by UV-visible spectroscopy based on the electronic state of the alkaline transition species of ferric and ferrous enzyme. The optical spectral data confirm an alkaline transition to hexacoordinate, low-spin heme species for native and wild-type MnP and show that the location of the engineered disulfide bonds in the protein can have significant effects on the electronic state of the enzyme. The addition of a single disulfide bond in the distal region of MnP resulted in an enzyme that maintained a pentacoordinate, high-spin heme at pH 9.0, whereas MnP with multiple engineered disulfide bonds did not exhibit an increase in stability of the pentacoordinate, high-spin state of the enzyme at alkaline pH. The mutant enzymes were assessed for increased stability by incubation at high pH. In comparison to wild-type MnP, enzymes containing engineered disulfide bonds in the distal and proximal regions of the protein retained greater levels of activity when restored to physiological pH. Additionally, when assayed for oxidase activity at pH 9.0, proteins containing engineered disulfide bonds exhibited slower rates of inactivation than wild-type MnP.
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Biotechnology Center, Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322-4705, USA.
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D000468:Alkalies / Q000737:chemistry; D002118:Calcium / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D004789:Enzyme Activation / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D005290:Ferric Compounds / Q000737:chemistry; D005296:Ferrous Compounds / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D010544:Peroxidases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D020075:Phanerochaete / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet
D000468:Alkalies; D004220:Disulfides; D005290:Ferric Compounds; D005296:Ferrous Compounds; D011994:Recombinant Proteins; D008345:Manganese; D010544:Peroxidases; C051129:manganese peroxidase; D002118:Calcium