Collins M J, Arciero D M, Hooper A B
Department of Genetics and Cell Biology, University of Minnesota, St. Paul 55108.
J Biol Chem. 1993 Jul 15;268(20):14655-62.
The hemes of hydroxylamine oxidoreductase (HAO) have been analyzed optically by potentiometric titrations using a low volume optically transparent thin layer electrochemical cell. The electrochemical behavior of the HAO monomeric unit has been interpreted by modeling the spectroelectrochemical data at several wavelengths to eight one-electron Nernst sites: seven c-type hemes and one P460 heme. Of the seven c-hemes, six show alpha-bands with absorption maxima at or near 553 nm. One c-heme has an alpha-band absorption maximum at 559 nm. The six c-553 hemes have midpoint potentials at pH 7.0 of +288, -10, -162, -192, -265 and -412 mV versus the normal hydrogen electrode (NHE). The c-559 heme has a midpoint potential (Em') at pH 7.0 of +11 mV versus NHE. The midpoint potential of the P460 heme is at -260 mV versus NHE at pH 7.0. In contrast, the midpoint potential for the P460 heme in another protein, cytochrome P460, from the same organism is -402 mV versus NHE at pH 7.0. Midpoint potentials of the c-hemes show little, if any, pH dependence over the range of pH 6-8. In contrast, Em' for the P460 heme changes with a slope of -60 mV/pH unit over the same range. Electrochemical isolation of the P460 heme at pH 8.0 led to the discovery of a broad spectroscopic feature centered near 740 nm that was assigned to the oxidized P460 heme. Changes in the spectroelectrochemical behavior of HAO after inactivation by H2O2 was almost exclusively restricted to the P460 heme of HAO. Both the 464-nm absorption band of the reduced P460 heme and the 740-nm band of the oxidized heme were no longer present. For the c-hemes, the only effect seems to be a slight shift in Em' for a single c-553 heme from -162 to -135 mV.
使用小体积光学透明薄层电化学池通过电位滴定法对羟胺氧化还原酶（HAO）的血红素进行了光学分析。通过对几个波长下的光谱电化学数据进行建模，将HAO单体单元的电化学行为解释为八个单电子能斯特位点：七个c型血红素和一个P460血红素。在七个c型血红素中，有六个显示出α带，其吸收最大值在553nm或接近553nm处。一个c型血红素的α带吸收最大值在559nm处。与标准氢电极（NHE）相比，六个c-553血红素在pH 7.0时的中点电位分别为+288、-10、-162、-192、-265和-412mV。c-559血红素在pH 7.0时相对于NHE的中点电位（Em'）为+11mV。P460血红素在pH 7.0时相对于NHE的中点电位为-260mV。相比之下，来自同一生物体的另一种蛋白质细胞色素P460中的P460血红素在pH 7.0时相对于NHE的中点电位为-402mV。在pH 6-8范围内，c型血红素的中点电位几乎没有pH依赖性（如果有也是很小）。相比之下，在相同范围内，P460血红素的Em'以-60mV/pH单位的斜率变化。在pH 8.0时对P460血红素进行电化学分离，发现了一个以740nm附近为中心的宽光谱特征，该特征被归因于氧化的P460血红素。H2O2使HAO失活后，HAO的光谱电化学行为变化几乎完全局限于HAO的P460血红素。还原的P460血红素的464nm吸收带和氧化血红素的740nm带都不再出现。对于c型血红素，唯一的影响似乎是单个c-553血红素的Em'从-162mV轻微偏移至-135mV。
