Engineering and Prosthetic‐Group Modification of Myoglobin: Peroxidase Activity, Chemical Stability and Unfolding Properties

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Abstract
The role of myoglobin (Mb) is not yet completely understood and recent evidence indicates it is involved in a variety of pseudo‐enzymatic functions. Mb is also extensively used as a tool to redesign novel active‐site features and introduce new activities into a protein. This review summarizes our approach to enhance the peroxidase‐like activity of Mb toward phenolic compounds by combining two different strategies of protein modification, i.e. active‐site engineering and cofactor replacement. The main objective of active‐site engineering is to increase the rate of hydrogen peroxide activation upon reaction at the iron(III) center, while cofactor replacement facilitates substrate interaction at the protein active site, thereby increasing the efficiency of the catalytic process and stereoselective recognition of the substrate. The thermodynamic stability of the modified Mb derivatives has been evaluated through guanidinium chloride induced and thermal unfolding experiments. As expected, both protein mutation and cofactor replacement decrease the stability of the protein, the latter effect being somewhat more pronounced for the Mb derivatives studied here. For evaluating the catalytic efficiency of the protein in non‐natural reactions, the chemical stability of the protein during catalysis is also important and therefore a new protocol was developed to evaluate the competitive degradation undergone by the protein upon substrate oxidation. It was found that autodegradation can occur through two different pathways involving oxidation of protein residues by the Mb active species or by a substrate‐derived phenoxy radical. The peroxidase‐like nitrating activity and the sulfoxidation reactions promoted by Mb, both involving the nitrite ion as a cosubstrate, have also been briefly described. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)