Functional Alteration of Myoglobin to Nitrite Reductase by Synthetic Heme and Protein Mutations

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In this project, we tried to alter the function of myoglobin, which serves as an oxygen storage in a muscle, to nitrite reductase, which reduces nitrite anion to nitric oxide in denitrification process. To prepare the artificial nitrite reductase from myoglobin, we first synthesized the active site model heme of the nitrite reductase. Since heme-d_1, which has been known as an active site of the nitrite reductase, has a dioxoisobacteriochlorin core, we prepared dioxoisobacterichlorin from dimethyldeutrioporphyrin with the oxidation by osmium tetraoxide and the pinocol rearrangement. The absorption spectrum of the synthesized heme-d_1 model was similar to that of nitrite reductase, suggesting the heme-d_1 model works as an active site of the artificial nitrite reductase. Therefore, we further tried to reconstitute the heme-d_1 model into apomyoglobin. The heme-d1 model formed a stable 1 : 1 complex with the apomyoglobin. The spectroscopic data of the complex were close to those of nitrite reductase. On the basis of these results, we further tried to add the protein mutations into the complex. We prepared L29H/F45H/H64Y myoglobin mutant because the structure of the mutant seems to be close to that of nitrite reductase. The mutant myoglobin was successfully expressed by e-coli and isolated. The triple mutant was also formed stable complex with heme-d1 model complex. The artificial nitrite reductase mimics not only the spectroscopic properties but also the reactivity of nitrite reducatse.