Department of Chemistry and Biochemistry, University of California at Santa Barbara, Santa Barbara, CA 93106-9510, USA.
J Inorg Biochem. 2012 Feb;107(1):47-53. doi: 10.1016/j.jinorgbio.2011.10.006. Epub 2011 Oct 25.
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(II)Mb and Co(II)Mb 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(II)Mb and Mn(II)Mb, respectively, both orders of magnitude slower than the k(2) (6M(-1)s(-1)) for wt Mb(II). The final reaction products for Mn(II)Mb consisted of a mixture of the nitrosyl Mn(II)Mb(NO) and Mn(III)Mb, similar to the products from the analogous NiR reaction by wt Mb. In contrast, the products of NiR by Co(II)Mb were found to be the nitrito complex Co(III)Mb(ONO(-)) plus roughly an equivalent of free NO. The differences can be attributed in part to the stronger coordination of inorganic nitrite to Co(III)Mb as reflected in the respective M(III)Mb(ONO(-)) formation constants K(nitrite): 2100 M(-1) (Co(III)) and <~0.4M(-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.
血红素蛋白将亚硝酸盐还原为一氧化氮，作为哺乳动物生理学中缺氧损伤的保护机制，正受到越来越多的关注。在这里，我们研究了锰（II）和钴（II）取代肌红蛋白的亚硝酸盐还原酶（NiR）活性，并与以前获得的铁（II）类似物 wt Mb（II）的数据进行了比较。Mn（II）Mb 和 Co（II）Mb 都表现出 NiR 活性，并且表明动力学在 [蛋白]、[亚硝酸盐]和 [H（+）] 中均为一级，如先前确定的 Fe（II）类似物 wt Mb（II）。在 pH 7.4 和 T=25°C 下，Co（II）Mb 和 Mn（II）Mb 的二级速率常数（k（2））分别为 0.0066 和 0.015 M（-1）s（-1），均比 wt Mb（II）的 k（2）（6M（-1）s（-1））慢几个数量级。Mn（II）Mb 的最终反应产物是混合的亚硝酰基 Mn（II）Mb（NO）和 Mn（III）Mb，类似于 wt Mb 类似的 NiR 反应产物。相比之下，Co（II）Mb 的 NiR 产物被发现是亚硝酰基配合物 Co（III）Mb（ONO（-））加上大约当量的游离 NO。差异部分归因于亚硝酸盐与 Co（III）Mb 的更强配位，这反映在各自的 M（III）Mb（ONO（-））形成常数 K（nitrite）中：2100 M（-1）（Co（III））和 <~0.4M（-1）（Mn（III））。我们还报告了突变体 metMb 的亚硝酸盐配合物 H64V Mb（III）（NO2（-））和 H64V/V67R Mb（III）（ONO（-））的形成常数（分别为 3.7 和 30 M（-1）），以及 wt metMb 的亚硝酸盐配合物的修订 K（nitrite）值（120 M（-1））。三个三价铁蛋白的相应 K（nitrite）值强调了氢键供体残基的重要性，例如 Mb（III）远端口袋中的 His64 或 H64V/V67R Mb（III）中的 Arg67，在稳定亚硝酸盐配位中。值得注意的是，相应亚铁 Mb 的 NiR 活性遵循相似的序列，表明亚硝酸盐与这些中心的结合同样受到氢键供体残基的影响。
