Nitrite reduction by CoII and MnII substituted myoglobins: Towards understanding necessary components of Mb nitrite reductase activity

Nitrite reduction to nitric oxide by heme proteins is drawing increasing attention as a protective mechanism to hypoxic injury in mammalian physiology. Here we probe the nitrite reductase (NiR) activities of manganese(II)- and cobalt(II)-substituted myoglobins, and compare with data obtained previously for the iron(II) analog wt MbII. Both MnIIMb and CoIIMb displayed NiR activity, and it was shown that the kinetics are first order each in [protein], [nitrite], and [H+], as previously determined for the FeII analog wt MbII. The second order rate constants (k2) at pH 7.4 and T = 25 °C, were 0.0066 and 0.015 M− 1 s− 1 for CoIIMb and MnIIMb, respectively, both orders of magnitude slower than the k2 (6 M− 1 s− 1) for wt MbII. The final reaction products for MnIIMb consisted of a mixture of the nitrosyl MnIIMb(NO) and MnIIIMb, similar to the products from the analogous NiR reaction by wt Mb. In contrast, the products of NiR by CoIIMb were found to be the nitrito complex CoIIIMb(ONO−) plus roughly an equivalent of free NO. The differences can be attributed in part to the stronger coordination of inorganic nitrite to CoIIIMb as reflected in the respective MIIIMb(ONO−) formation constants Knitrite: 2100 M− 1 (CoIII) and <~0.4 M− 1 (MnIII). We also report the formation constants (3.7 and 30 M− 1, respectively) for the nitrite complexes of the mutant metmyoglobins H64V MbIII(NO2−) and H64V/V67R MbIII(ONO−) and a Knitrite revised value (120 M− 1) for the nitrite complex of wt metMb. The respective Knitrite values for the three ferric proteins emphasize the importance of a H-bonding residue, such as His64 in the MbIII distal pocket or the Arg67 in H64V/V67R MbIII, in stabilizing nitrite coordination. Notably, the NiR activities of the corresponding ferrous Mbs follow a similar sequence suggesting that nitrite binding to these centers are analogously affected by the H-bonding residues.

Graphical abstractNitrite reduction to NO by wt Mb(II) and its metal-substituted compounds follows the order wt Mb ≫ MnMb > CoMb.Figure optionsDownload full-size imageDownload as PowerPoint slideHighlights► The conversion of nitrite to NO by Mn- and Co-substituted myoglobins was probed. ► The Mb-nitrite formation constants follow the order Co(III)Mb > Fe(III)Mb ≫ Mn(III)Mb. ► Both Mn(II)Mb and Co(II)Mb display nitrite reductase (NiR) reactivity. ► The order of NiR reactivity follows the order wt Mb(II) ≫ Mn(II)Mb > Co(II)Mb.