Rational design of functional heme copper oxidases in myoglobin

We redesign an existing well-studied heme protein, myoglobin, to mimic the bimetallic, heme-CuB site of heme copper oxidases (HCOs). This is the site where molecular oxygen is converted to water as part of cellular respiration. The conversion of oxygen to water is highly difficult as there are many highly reactive intermediates that must stabilized so that the reaction can result in water formation. Such a redesign can be thought of as going from the "bottom up" with respect to the desired function. In the process of building up such a model, we are producing minimalistic versions in order to see what the function of each of the structural features is and how it affects chemistry.;This thesis describes the improvement of an existing myoglobin based model system of HCOs, named CuBMb, where an non-native copper site was previously engineered into myoglobin by adding two histidine residues. Along with the native histidine, the resulting site resembles the Cu B site found in HCOs. This model protein is purified without metal in the CuB site and therefore it is possible to determine the role of the bound metal and the effect of using other metals. Previous studies of this model have not observed the desired chemistry, production of water from oxygen. However, HCOs have a novel feature found in no other proteins, namely a covalently attached histidine and tyrosine moiety that is critical for function of HCOs in vivo. To roughly mimic this novel feature, a tyrosine was introduced into CuBMb at various locations in the designed heme-CuB site. To guide the selection of the positions to place our tyrosine we used both the amino acid sequence information of HCOs and computer based protein models of myoglobin with a tyrosine. The computer models were compared to reported crystal structures of HCOs. The most similar mutants were made and characterized. The resulting tyrosine containing Cu BMbs displayed the ability to produce water from oxygen, despite the absence of the covalent bond between tyrosine and one of the hisitidines used to bind the copper, as in HCOs. Even more unexpectedly the desired activity was observed without the copper in the CuB site. This result is both interesting and unexpected. To futher improve the observed rate, more features of HCOs such as proton delivery channels and non-natural heme cofactors with similar features compared to heme cofactors found in HCOs were introduced into myoglobin. The proton channels had positive effects on the observed activity. In addition to these interesting results, attempts were made to try and react the tyrosine containing CuBMbs under various conditions to induce formation of a covalent bond analogous to the crosslinked histidine and tyrosine found in HCOs. In the process, a crystal structure of a novel species, where an oxygen species is bound "side-on" to the metal of the heme cofactor instead of the expected "end-on" mode that to our knowledge has never been observed in a heme protein.;In summary, to better understand the functioning complex proteins like HCOs, a model protein was previously constructed. Introduction of new structural features, into the model protein (CuBMb), with the purpose of mimicking features similar to those found in found in HCOs caused the model system to become competent to perform the desired chemistry with less features than what is thought to be required in HCOs. As more features were attempted we discovered an interesting oxygen species bound to our protein. These type of results show the advantage of trying to build upto a minimal model. It is possible with such a system to obtain unique proteins and intermediates in addition to what information one is attempting elucidate. One is also able to perform experiments that would be impossible in the native system and obtain useful information. The insights gained by this modeling work will help the designers of the next version of CuBMb overcome the limitations of this version and gain insight into how to generally build and design metalloproteins. (Abstract shortened by UMI.).