Role of disulfide bonds in the stability of recombinant manganese peroxidase

Phanerochaete chrysosporium manganese peroxidase (MnP) [isoenzyme H4] was e ngineered with additional disulfide bonds to provide structural reinforceme nt to the proximal and distal calcium-binding sites. This rational protein engineering investigated the effects of multiple disulfide bonds on the sta bilization of the enzyme heme environment and oxidase activity. Stabilizati on of the heme environment was monitored by UV-visible spectroscopy based o n the electronic state of the alkaline transition species of ferric and fer rous enzyme. The optical spectral data confirm an alkaline transition to he xacoordinate, low-spin heme species for native and wild-type MnP and show t hat 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 t hat maintained a pentacoordinate, high-spin heme at pH 9.0. whereas MnP wit h multiple engineered disulfide bonds did not exhibit an increase in stabil ity of the pentacoordinate, high-spin state of the enzyme at alkaline pH. T he mutant enzymes were assessed for increased stability by incubation at hi gh pH. In comparison to wild-type MnP, enzymes containing engineered disulf ide bonds in the distal and proximal regions of the protein retained greate r levels of activity when restored to physiological pH. Additionally, when assayed for oxidase activity at pH 9.0, proteins containing engineered disu lfide bonds exhibited slower rates of inactivation than wildtype MnP.