Roles of Two Interhelical Insertions in Catalase-peroxidase Catalysis: Tracing the Impact of Peripheral Protein Structures on Heme Enzyme Function

Abstract

Monofunctional peroxidases and bifunctional catalase-peroxidases share almost superimposable active sites, yet peroxidases lack appreciable catalase activity. Moreover, catalase-peroxidases catalyze both catalase and peroxidase reactions with a single active site. Given that catalase-peroxidases have two extra interhelical insertions that connect the D and E helices (the DE insertion), and F and G helices (the FG insertion), it is rational to suggest that these two insertions may serve to fine-tune the active site for bifunctionality. To explore the roles of these two insertions, we produced variants of E. coli catalase-peroxidase (KatG) lacking either the DE insertion (KatG?DE), the FG insertion (KatG?FG), or both insertions (KatG?DE/FG). All variants retained peroxidase activity that was comparable or greater than that of wtKatG. However, KatG?FG retained only 0.2% catalase activity, whereas KatG?DE and KatG?DE/FG lost all catalase activity, indicating both insertions are critical for KatG bifunctional ability. KatG?FG absorption and EPR spectra suggested little change in heme coordination state occurred when FG insertion was removed. Kinetic parameters suggested that the FG insertion poises the active site geometrically or electronically to help peroxide substrates access to the active site. In contrast, KatG?DE appeared to have more hexa-coordinate heme species present, suggesting this insertion is important for maintaining the correct heme coordination environment. The kinetic parameters suggest that the DE insertion serves to regulate the access of reducing substrates to the heme edge. As a result, the rate limiting step of the KatG?DE peroxidase catalytic cycle changed from compound I reduction as for wtKatG to compound II reduction. This led to an increased propensity to the formation of compound III for KatG?DE. This phenomenon is also commonly observed in monofunctional peroxidases. This inactivation could be prevented by the reducing substrate due to its competition with H2O2 in reacting with compound II. Furthermore, the inactive compound III could also be rescued back to the catalytic cycle by reducing substrate cation radicals. Both insertions, despite of their peripheral positions to the active site, serve to fine-tune the active site for its bifunctional properties and cooperate with other peripheral protein structures to achieve the full catalytic potential of the catalase-peroxidases.