Novel Redox Active Tyrosine Mutations Enhance the Regeneration of Functional Oxyhemoglobin from Methemoglobin: Implications for Design of Blood Substitutes.

G G A Silkstone, M Simons, B S Rajagopal, T Shaik, B J Reeder, C E Cooper

Advances in experimental medicine and biology 2018

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Heme mediated oxidative toxicity has been linked to adverse side effects in Hemoglobin Based Oxygen Carriers (HBOC), initiated by reactive ferryl (FeIV) iron and globin based free radical species. We recently showed that the addition of a redox active tyrosine residue in the beta subunit (βF41Y) of recombinant hemoglobin had the capability to decrease lipid peroxidation by facilitating the reduction of FeIV iron by plasma antioxidants such as ascorbate. In order to explore this functionality further we created a suite of tyrosine mutants designed to be accessible for both reductant access at the protein surface, yet close enough to the heme cofactor to enable efficient electron transfer to the FeIV. The residues chosen were: βF41Y; βK66Y; βF71Y; βT84Y; βF85Y; and βL96Y. As with βF41Y, all mutants significantly enhanced the rate of ferryl (FeIV) to ferric (FeIII) reduction by ascorbate. However, surprisingly a subset of these mutations (βT84Y, and βF85Y) also enhanced the further reduction of ferric (FeIII) to ferrous (FeII) heme, regenerating functional oxyhemoglobin. The largest increase was seen in βT84Y with the percentage of oxyhemoglobin formed from ferric hemoglobin in the presence of 100 μM ascorbate over a time period of 60 min increasing from 10% in βF41Y to over 50% in βT84Y. This increase was accompanied by an increased rate of ascorbate consumption. We conclude that the insertion of novel redox active tyrosine residues may be a useful component of any recombinant HBOC designed for longer functional activity without oxidative side effects.

Citation

G G A Silkstone, M Simons, B S Rajagopal, T Shaik, B J Reeder, C E Cooper. Novel Redox Active Tyrosine Mutations Enhance the Regeneration of Functional Oxyhemoglobin from Methemoglobin: Implications for Design of Blood Substitutes. Advances in experimental medicine and biology. 2018;1072:221-225