Rifkind J M, Lauer L D, Chiang S C, Li N C
Biochemistry. 1976 Nov 30;15(24):5337-43. doi: 10.1021/bi00669a021.
Oxidation studies of hemoglobin by Cu(II) indicate that for horse hemoglobin, up to a Cu(II)/heme molar ratio of 0.5, all of the Cu(II) added is used to rapidly oxidize the heme. On the other hand, most of the Cu(II) added to human hemoglobin at low Cu(II)/heme molar ratios is unable to oxidize the heme. Only at Cu(II)/heme molar ratios greater than 0.5 does the amount of oxidation per added Cu(II) approach that of horse hemoglobin. At the same time, binding studies indicate that human hemoglobin has an additional binding site involving one copper for every two hemes, which has a higher copper affinity than the single horse hemoglobin binding site. The Cu(II) oxidation of human hemoglobin is explained utilizing this additional binding site by a mechanism where a transfer of electrons cannot occur between the heme and the Cu(II) bound to the high affinity human binding site. The electron transfer must involve the Cu(II) bound to the lower affinity human hemoglobin binding site, which is similar to the only horse hemoglobin site. The involvement of beta-2 histidine in the binding of this additional copper is indicated by a comparison of the amino acid sequences of various hemoglobins which possess the additional site, with the amino acid sequences of hemoglobins which do not possess the additional site. Zn(II), Hg(II), and N-ethylmaleimide (NEM) are found to decrease the Cu(II) oxidation of hemoglobin. The sulfhydryl reagents, Hg(II) and NEM, produce a very dramatic decrease in the rate of oxidation, which can only be explained by an effect on the rate for the actual transfer of electrons between the Cu(II) and the Fe(II). The effect of Zn(II) is much smaller and can, for the most part, be explained by the increased oxygen affinity, which affects the ligand dissociation process that must precede the electron transfer process.
铜（II）对血红蛋白的氧化研究表明，对于马血红蛋白，在铜（II）/血红素摩尔比高达0.5时，所有添加的铜（II）都用于快速氧化血红素。另一方面，在低铜（II）/血红素摩尔比下添加到人类血红蛋白中的大部分铜（II）无法氧化血红素。只有在铜（II）/血红素摩尔比大于0.5时，每添加一个铜（II）的氧化量才接近马血红蛋白的氧化量。同时，结合研究表明，人类血红蛋白还有一个额外的结合位点，每两个血红素涉及一个铜，该位点对铜的亲和力高于单一的马血红蛋白结合位点。人类血红蛋白的铜（II）氧化是利用这个额外的结合位点来解释的，其机制是电子无法在血红素和与高亲和力人类结合位点结合的铜（II）之间转移。电子转移必须涉及与低亲和力人类血红蛋白结合位点结合的铜（II），该位点与唯一的马血红蛋白位点相似。通过比较具有额外位点的各种血红蛋白的氨基酸序列与不具有额外位点的血红蛋白的氨基酸序列，表明β-2组氨酸参与了这种额外铜的结合。发现锌（II）、汞（II）和N-乙基马来酰亚胺（NEM）会降低血红蛋白的铜（II）氧化。巯基试剂汞（II）和NEM会使氧化速率大幅降低，这只能通过对铜（II）和铁（II）之间实际电子转移速率的影响来解释。锌（II）的影响要小得多，在很大程度上可以通过增加的氧亲和力来解释，氧亲和力会影响电子转移过程之前必须发生的配体解离过程。
