Characterization and design of hydrogen bonding interactions in oxygen reduction by engineered myoglobins

Proteins participate in nearly every function of living cells, and an estimated 50% are associated with a metal ion or cofactor. Most, if not all, of the fundamental chemical processes necessary for life, including photosynthesis and respiration, are facilitated by one or more metalloenzymes or metalloproteins. Metal-nucleic acid interactions are also clinically important in cancer and infection treatment, and are useful in industrial purposes. The relationship between macromolecule and metal can almost be viewed as symbiotic – metals often function at extreme limits when free in solution, with minimal ligands; on the other had, amino and nucleic acid polymers have limited functional capabilities. By capturing and precisely holding metal ions, biomolecules tame their reactivities by tuning their electronic and geometric – and therefore functional – properties, allowing the metal and protein together to perform precise chemical functions. However, despite decades of research, the intricate details of how this precision is imparted, in each case and more generally, are not entirely understood. In particular, the roles of long-range (beyond 1-2 bonds), non-covalent interactions are understood to be important; however, exactly how they impart function, is difficult to disentangle, due to the delicate balance resulting from evolution. Moreover, many of these systems, such as the oxidases and photosystems responsible for respiration and photosynthesis, respectively, are large, difficult to purify, and contain multiple cofactors that complicate investigations of the active site chemistry. Therefore, in the Lu lab, we have been pioneers of the "biosynthetic" modeling approach, in which the active sites of complex enzymes are structurally reconstructed in small, experimentally tractable proteins. In this vein, the active site of heme-copper oxidases (HCOs) – a bimetallic heme-copper center known as CuB – was previously structurally modeled in sperm whale myoglobin, by introducing two additional histidine residues, giving a protein called CuBMb. Further structural modeling of this protein to include additional structural features, such as a tyrosine residue, brought about not only partial HCO-like oxygen reduction activity, but an unexpected observation – the Cu ion was found to not be necessary for imparting this function, nor did it seem to improve it, in the designed protein. On the other hand the presence and positioning of the tyrosine had a dramatic effect on this activity. This thesis describes efforts to first understand the reaction of these CuBMbs with oxygen. It is found that the extended hydrogen bonding network, stabilized by the introduced residues and consisting of water molecules, is critical for imparting oxygen reduction activity to an oxygen binding protein. Based on this observation, further designs are pursued to improve this hydrogen-bonding network, in an effort to improve activity. It is found that incorporation of a glutamate within hydrogen bonding distance of one of the water molecules composing the extended hydrogen bonding network improves the function of the enzyme by eliminating the ROS release. Moreover, spectroscopic and crystallographic evidence support that this improvement is due to enhanced hydrogen bonding and protonation to the oxygen intermediate. This design demonstrates the critical importance of long-range interactions in enzymes, and should inspire enzyme engineers to pursue incorporation of these types of features. In addition to these studies, this thesis also reports the computational design of a novel heterobimetallic heme-Fe4S4 site, and progress in obtaining the first crystal structure of a metal mediated catalytic DNA construct