The overall goal of the project is to answer a key scientific question in the fields of bioenergetics and the nitrogen cycle, i.e., why a protein is proficient at O-O bond cleavage when using a heme-copper center as in heme-copper oxidases (HCO), and is effective at N-N bond formation when using a heme-non-heme iron center, as in nitric oxide reductase (NOR). We seek to overcome a critical methodological barrier to progress in this field by developing a novel biosynthetic approach that utilizes a stable, easy-to-produce, and well-characterized heme protein (myoglobin) as a scaffold for making structural and functional models of HCO and NOR. Building on the success of the last grant period when both CuB and FeB sites were designed into Mb (called CuBMb and FeBMb, respectively), as demonstrated by both spectroscopy and X-ray crystallography, site-directed mutagenesis and expressed protein ligation (EPL) will be employed to introduce natural or unnatural amino acids into the metal-binding sites. Studies of the model proteins will be carried out using spectroscopy as well as X-ray crystallography. Kinetic studies will also be performed to provide insight into the mechanisms of O2 and NO reduction. This proposal contains three specific aims that our biosynthetic approach can uniquely address when compared to other approaches: 1) elucidate structural features responsible for HCO function, specifically the roles of CuB site, the tyrosine that is covalently linked to a CuB-His ligand, and non-covalent interactions, such as water and the associated hydrogen bonding network;2) elucidate structural features responsible for NOR function, specifically the FeB site and nearby glutamate residues;and 3) examine structural and functional differences between the heme-copper center in HCO and the heme-non-heme iron center in NOR. Once the project aims are achieved, it will result in a deeper understanding of the structure and function of HCO and NOR. The placement of a heme-copper and a heme- iron center in the same protein framework, and the ability to introduce different metal ions into the CuB or FeB sites in the same protein is a unique opportunity that allows direct structural and functional comparisons of two important classes of metal-binding centers. At the same time, the project will advance our knowledge of heme protein structure, function and design in general, as the guiding principles obtained from the studies may be applicable to a broad range of other metalloproteins.
The proposed research is relevant to health in a broad sense. HCOs are the terminal electron acceptors in the respiratory chain of all aerobic organisms and are required for aerobic life. Furthermore, deficiencies of or mutations in HCOs have been linked to Alzheimer's disease, Leigh syndrome and aging. Well-studied denitrification enzymes such as NOR may provide potential structural and spectroscopic models for mammalian enzymes that produce and utilize NO in a variety of signal transduction pathways. Therefore the work will make important contributions to healthcare, as it will provide a molecular basis for understanding two enzymes important to human health.
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|Sabuncu, Sinan; Reed, Julian H; Lu, Yi et al. (2018) Nitric oxide reductase activity in heme-nonheme binuclear engineered myoglobins through a one-electron reduction cycle. J Am Chem Soc :|
|Lu, Yi (2017) The ""OK, Molly"" Chemistry. Acc Chem Res 50:647-651|
|Reed, Julian H; Shi, Yelu; Zhu, Qianhong et al. (2017) Manganese and Cobalt in the Nonheme-Metal-Binding Site of a Biosynthetic Model of Heme-Copper Oxidase Superfamily Confer Oxidase Activity through Redox-Inactive Mechanism. J Am Chem Soc 139:12209-12218|
|Bhagi-Damodaran, Ambika; Michael, Matthew A; Zhu, Qianhong et al. (2017) Why copper is preferred over iron for oxygen activation and reduction in haem-copper oxidases. Nat Chem 9:257-263|
|Bhagi-Damodaran, Ambika; Kahle, Maximilian; Shi, Yelu et al. (2017) Insights Into How Heme Reduction Potentials Modulate Enzymatic Activities of a Myoglobin-based Functional Oxidase. Angew Chem Int Ed Engl 56:6622-6626|
|Bhagi-Damodaran, Ambika; Petrik, Igor; Lu, Yi (2016) Using Biosynthetic Models of Heme-Copper Oxidase and Nitric Oxide Reductase in Myoglobin to Elucidate Structural Features Responsible for Enzymatic Activities. Isr J Chem 56:773-790|
|Petrik, Igor D; Davydov, Roman; Ross, Matthew et al. (2016) Spectroscopic and Crystallographic Evidence for the Role of a Water-Containing H-Bond Network in Oxidase Activity of an Engineered Myoglobin. J Am Chem Soc 138:1134-7|
|Matsumura, Hirotoshi; Chakraborty, Saumen; Reed, Julian et al. (2016) Effect of Outer-Sphere Side Chain Substitutions on the Fate of the trans Iron-Nitrosyl Dimer in Heme/Nonheme Engineered Myoglobins (Fe(B)Mbs): Insights into the Mechanism of Denitrifying NO Reductases. Biochemistry 55:2091-9|
|Bhagi-Damodaran, A; Hosseinzadeh, P; Mirts, E et al. (2016) Design of Heteronuclear Metalloenzymes. Methods Enzymol 580:501-37|
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