Functional diversity of myoglobin isozymes from Carassius auratus - Biophysical study of myoglobin as a peroxidase

Myoglobin (Mb) is a heme protein expressed in oxidative muscle tissue and functions in oxygen storage and transport. Mb can also display complex redox chemistry involved in several important physiological processes including scavenging of reactive oxygen species and nitric oxide metabolism. Goldfish and the common carp are the only vertebrates known today that express two paralogs of Mb (Mb1 and Mb2) as a result of a recent whole genome duplication. Mb1 is expressed in gills, liver, and skeletal muscle tissue, whereas Mb2 is exclusively expressed in the brain. The tissue-specific expression led to work showing that Mb2 in carp fish is a more effective peroxidase. In this study, work has been done to gain detailed structural and functional insight into the differences of the goldfish Mb1 and Mb2 isozymes by biophysical characterization and 3D structure analysis. The intensively studied Mb from horse heart (hMb) has been studied together with Mb1 and Mb2 as a reference. The reaction of Mb with hydrogen peroxide (H2O2) has been of particular interest. In addition, studies on how radiation damage during diffraction data collection affects redox states of Mb have been performed. This study shows that goldfish Mb2 reacts with H2O2 significantly faster than Mb1. Structural modeling of Mb1 and Mb2 indicates that no differences around the active site are responsible for the difference in reactivity. The structural basis of this functional difference is therefore complex in nature and might be explained by altered dynamics as a consequence of differing amino acid composition towards the surface of the proteins. The ferric and ferryl redox states of Mb are damaged by X-ray radiation during diffraction data collection. The ferric state is significantly more susceptible to radiation damage compared to the ferryl state. This study shows the importance of performing a time/dose calculation on the amount of radiation a specific redox state can handle when collecting diffraction data