Max-Planck-Institut für Bioanorganische Chemie, Stiftstrasse 34-36, D-45470 Mülheim an der Ruhr, Germany.
Biochemistry. 2010 Jul 20;49(28):5841-51. doi: 10.1021/bi100324z.
The interaction of ferriheme proteins with nitrite has recently attracted interest as a source for NO or other nitrogen oxides in mammalian physiology. However, met-hemoglobin (metHb), which was suggested as a key player in this process, does not convert nitrite unless small amounts of NO are added in parallel. We have recently reported that, in contrast, nitrophorins (NPs) convert nitrite as the sole substrate to form NO even at pH 7.5, which is an unprecedented case among ferrihemes [He, C., and Knipp, M. (2009) J. Am. Chem. Soc. 131, 12042-12043]. NPs, which comprise a class of unique heme b proteins from the saliva of the blood-sucking insect Rhodnius prolixus, appear in a number of concomitant isoproteins. Herein, the first spectroscopic characterization of the initial complexes of the two isoproteins NP4 and NP7 with nitrite is presented and compared to the data reported for metHb and met-myoglobin (metMb). Because upon nitrite binding, NPs, in contrast to metHb and metMb, continue to react with nitrite, resonance Raman spectroscopy and continuous wave electron paramagnetic resonance spectroscopy were applied to frozen samples. As a result, the existence of two six-coordinate ferriheme low-spin complexes was established. Furthermore, X-ray crystallography of NP4 crystals soaked with nitrite revealed the formation of an eta(1)-N nitro complex, which is in contrast to the eta(1)-O-bound nitrite in metMb and metHb. Stopped-flow kinetic experiments show that although the ligand dissociation constants of NP4 and NP7 (15-190 M(-1)) are comparable to those of metHb and metMb, the rates of ligand binding and release are significantly slower. Moreover, not only the reaction kinetics but also electron paramagnetic resonance spectroscopy reveals notable differences between the two isoproteins.
亚铁血红素蛋白与亚硝酸盐的相互作用最近引起了人们的兴趣，因为它可能是哺乳动物生理学中 NO 或其他氮氧化物的来源。然而，高铁血红蛋白（metHb）被认为是该过程中的关键因素，但除非同时添加少量的 NO，否则它不会将亚硝酸盐转化为其他物质。我们最近报道称，相反，硝普蛋白（NPs）可以将亚硝酸盐作为唯一的底物转化为 NO，即使在 pH 值为 7.5 的情况下也是如此，这在亚铁血红素中是前所未有的[He，C.和 Knipp，M.（2009）J. Am. Chem. Soc. 131, 12042-12043]。NPs 是来自吸血昆虫 Rhodnius prolixus 唾液的一类独特的血红素 b 蛋白，由许多同工蛋白组成。本文首次报道了两种同工蛋白 NP4 和 NP7 与亚硝酸盐形成的初始复合物的光谱特征，并与 metHb 和 met-myoglobin（metMb）的相应数据进行了比较。因为与 metHb 和 metMb 不同，NPs 在与亚硝酸盐结合后会继续与亚硝酸盐反应，所以共振拉曼光谱和连续波电子顺磁共振光谱被应用于冷冻样品。结果表明，存在两种六配位亚铁血红素低自旋配合物。此外，NP4 晶体与亚硝酸盐浸泡后的 X 射线晶体学揭示了形成 eta(1)-N 硝基配合物，这与 metMb 和 metHb 中的 eta(1)-O 结合的亚硝酸盐形成对比。停流动力学实验表明，尽管 NP4 和 NP7 的配体解离常数（15-190 M(-1))与 metHb 和 metMb 的配体解离常数相当，但配体结合和释放的速率明显较慢。此外，不仅反应动力学，而且电子顺磁共振光谱也揭示了两种同工蛋白之间的显著差异。
