Design of Heteronuclear Metalloenzymes

Abstract

Heteronuclear metalloenzymes catalyze some of the most fundamentally interesting and practically useful reactions in nature. However, the presence of two or more metal ions in close proximity in these enzymes makes them more difficult to prepare and study than homonuclear metalloenzymes. To meet these challenges, heteronuclear metal centers have been designed into small and stable proteins with rigid scaffolds to understand how these heteronuclear centers are constructed and the mechanism of their function. This chapter describes methods for designing heterobinuclear metal centers in a protein scaffold by giving specific examples of a few heme-nonheme bimetallic centers engineered in myoglobin and cytochrome c peroxidase. We provide step-by-step procedure on how to choose the protein scaffold, design a heterobinuclear metal center in the protein computationally, incorporate metal centers in the protein and characterize the resulting metalloprotein, both structurally and functionally. Finally, we discuss how an initial design can be further improved by rationally tuning its secondary coordination sphere, electron/proton transfer rates, and the substrate affinity.

Artificial Heme Enzymes for the Construction of Gold-Based Biomaterials

“…Extensive progress in protein design and engineering enables the construction of artificial metalloenzymes showing efficiency and substrate diversity beyond those of natural systems [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 ]. Heme-containing enzymes active in oxidation chemistry are a fascinating source of inspiration for researchers [ 13 ].…”

Enzymes and their turnover numbers

“…Turnover number defines the rate at which an enzyme converts its substrate, usually in terms of the number of substrate molecules that can be converted by a single enzyme molecule and can range from a few molecules to several million molecules per second [1].…”

Understanding and Modulating Metalloenzymes with Unnatural Amino Acids, Non-Native Metal Ions, and Non-Native Metallocofactors

“…2C). 34 In addition, the rate of O 2 reduction and enzyme turnover number were both positively correlated with heme Eº´ while ROS production remained essentially constant. Rapid stopped-flow kinetics studies found that increased heme Eº´ was associated with increases in the rates of ET, O2 binding, O 2 dissociation, and a slight decrease in O 2 affinity.…”

“…When we replaced the nonheme Fe(II) in Fe B Mb with Cu(I), O 2 reduction activity increased 3-fold. 55 A further study to explain this effect found that the identity of the nonheme metal had minimal impact on heme Eº´, but Eº´ of Cu(II)/Cu(I) is +128 mV higher than nonheme Fe(II)/Fe(III). Since the rate of O 2 reduction is limited by ET, a stronger driving force for ET to bound O 2 is partially responsible for the observed rate enhancement with Cu.…”