Engineering tyrosine electron transfer pathways decreases oxidative toxicity in hemoglobin: implications for blood substitute design.

Hemoglobin (Hb)-based oxygen carriers (HBOC) have been engineered to replace or augment the oxygen-carrying capacity of erythrocytes. However, clinical results have generally been disappointing due to adverse side effects linked to intrinsic heme-mediated oxidative toxicity and nitric oxide (NO) scavenging. Redox-active tyrosine residues can facilitate electron transfer between endogenous antioxidants and oxidative ferryl heme species. A suitable residue is present in the α-subunit (Y42) of Hb, but absent from the homologous position in the ß-subunit (F41). We therefore replaced this residue with a tyrosine (ßF41Y, Hb Mequon). The ßF41Y mutation had no effect on the intrinsic rate of lipid peroxidation as measured by conjugated diene and singlet oxygen formation following the addition of ferric(met) Hb to liposomes. However, ßF41Y significantly decreased these rates in the presence of physiological levels of ascorbate. Additionally, heme damage in the ß-subunit following the addition of the lipid peroxide hydroperoxyoctadecadieoic acid was five-fold slower in ßF41Y. NO bioavailability was enhanced in ßF41Y by a combination of a 20% decrease in NO dioxygenase activity and a doubling of the rate of nitrite reductase activity. The intrinsic rate of heme loss from methemoglobin was doubled in the ß-subunit, but unchanged in the α-subunit. We conclude that the addition of a redox-active tyrosine mutation in Hb able to transfer electrons from plasma antioxidants decreases heme-mediated oxidative reactivity and enhances NO bioavailability. This class of mutations has the potential to decrease adverse side effects as one component of a HBOC product.

Nitrogen dioxide oxidizes mitochondrial cytochrome c.

We previously reported that high micromolar concentrations of nitric oxide were able to oxidize mitochondrial cytochrome c at physiological pH, producing nitroxyl anion (Sharpe and Cooper, 1998 Biochem. J. 332, 9-19). However, the subsequent re-evaluation of the redox potential of the NO/NO(-) couple suggests that this reaction is thermodynamically unfavored. We now show that the oxidation is oxygen-concentration dependent and non stoichiometric. We conclude that the effect is due to an oxidant species produced during the aerobic decay of nitric oxide to nitrite and nitrate. The species is most probably nitrogen dioxide, NO(2)(•) a well-known biologically active oxidant. A simple kinetic model of NO autoxidation is able to explain the extent of cytochrome c oxidation assuming a rate constant of 3×10(6)M(-1)s(-1) for the reaction of NO(2)(•) with ferrocytochrome c. The importance of NO(2)(•) was confirmed by the addition of scavengers such as urate and ferrocyanide. These convert NO(2)(•) into products (urate radical and ferricyanide) that rapidly oxidize cytochrome c and hence greatly enhance the extent of oxidation observed. The present study does not support the previous hypothesis that NO and cytochrome c can generate appreciable amounts of nitroxyl ions (NO(-) or HNO) or of peroxynitrite.