Nitrite reduction by Co II and Mn II substituted myoglobins : Towards understanding necessary components of Mb nitrite reductase activity

Abstract

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 Mb II. Both Mn IIMb and Co IIMb displayed NiR activity, and it was shown that the kinetics are first order each in [protein], [nitrite], and [H +], as previously determined for the Fe II analog wt Mb II. The second order rate constants ( k 2 ) at pH 7.4 and T = 25 °C, were 0.0066 and 0.015 M − 1 s − 1 for Co IIMb and Mn IIMb, respectively, both orders of magnitude slower than the k 2 (6 M − 1 s − 1 ) for wt Mb II. The final reaction products for Mn IIMb consisted of a mixture of the nitrosyl Mn IIMb(NO) and Mn IIIMb, similar to the products from the analogous NiR reaction by wt Mb. In contrast, the products of NiR by Co IIMb were found to be the nitrito complex Co IIIMb(ONO −) plus roughly an equivalent of free NO. The differences can be attributed in part to the stronger coordination of inorganic nitrite to Co IIIMb as reflected in the respective M IIIMb(ONO −) formation constants K nitrite : 2100 M − 1 (Co III) and <~0.4 M − 1 (Mn III). We also report the formation constants (3.7 and 30 M − 1 , respectively) for the nitrite complexes of the mutant metmyoglobins H64V Mb III(NO 2 −) and H64V/V67R Mb III(ONO −) and a K nitrite revised value (120 M − 1 ) for the nitrite complex of wt metMb. The respective K nitrite values for the three ferric proteins emphasize the importance of a H-bonding residue, such as His64 in the Mb III distal pocket or the Arg67 in H64V/V67R Mb III, 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.