From Myoglobin to Heme-Copper Oxidase: Design and Engineering of a CuB Center into Sperm Whale Myoglobin

Abstract

Myoglobins (Mb) are small globular heme proteins that serve as O2 carriers. Heme-copper oxidases (HCOs) are large membrane-bound proteins that catalyze proton-coupled reduction of O2 to water. Mb contains a single heme center, while HCOs contain a high-spin heme-CuB dinuclear center, a low-spin heme center, and in certain subclasses of HCO enzymes such as cytochrome c oxidases (CcO) a dinuclear copper center called CuA. While Mb is one of the most well-characterized proteins, many questions about the structure and function of HCOs, such as the role of the CuB center, the origin of spin coupling between CuB and heme, and the exact nature of the reaction intermediates, remain to be fully understood. We report here the design and engineering of a copper-binding site in sperm whale myoglobin (swMb) based on structural comparison and computer modeling of swMb and CcO. UV−vis studies of the resting state of the designed protein swMb((L29H, F43H) (called CuBMb) suggest that a single copper-binding site is created in swMb. UV−vis, elemental analysis, and EPR studies of the cyanide-bound CuBMb indicate that a spin-coupled, CN--bridged CuB-heme center is formed in the designed model protein, as in the native HCOs. Parallel spectroscopic studies with Zn(II) in the place of Cu(II) further support the conclusion. The study also reveals that the presence of Cu(II) and Ag(I) (as a Cu(I) mimic) increased the affinity of heme for diatomic ligands such as CN- and O2. This study shows that it is possible to design and engineer metal-binding sites in proteins with little sequence and structural homology. The resulting designed protein, free from other chromophores, is more amendable to biochemical and biophysical studies. Spectroscopy studies of the designed protein indicate that the CuB center plays an important role in HCO structure and function.