Hershberg R D, Chance B
Biochemistry. 1975 Aug 26;14(17):3885-91. doi: 10.1021/bi00688a023.
The binding of formate ion, a substrate for the peroxidatic reaction of catalase, has been investigated by magnetic resonance techniques. Comparative studies of formate binding to ferric myoglobin have also been performed. The nuclear magnetic relaxation (NMR) rate of formate and water protons is enhanced by the presence of ferric horse liver catalase. The enhancement is not changed significantly by the addition of cyanide, indicating that water and formate are still bound in the presence of cyanide. Formate proton to heme iron distances determined by magnetic resonance techniques indicate that formate does not directly bind to the heme iron of catalase or myoglobin but to the globin, and NMR relaxation occurs as a result of outersphere mechanisms. Evidence that water forms an innersphere complex with the iron atom of the catalase heme is presented. In similar experiments with ferric myoglobin, the addition of cyanide caused a large decrease in the enhancement of the proton relaxation rate of both formate and water, indicating the displacement of water and formate from the heme and the vicinity of the heme, respectively. Broad, high-spin, ferric ion electron paramagnetic resonance absorptions of catalase and myoglobin at room temperature obtained in the presence and absence of formate show that formate does not alter appreciably the heme environment of catalase or myoglobin or the spin state of the heme iron. Studies on the binding of formate to catalase as monitored by changes in the heme absorption spectrum in the visible region show one-to-one stoichiometry with heme concentration. However, the small changes observed in the visible region of the optical spectrum on addition of formate ion are attributed to a secondary effect of formate on the heme environment, rather than direct binding of formate to the heme moiety.
过氧化氢酶过氧化物反应的底物甲酸根离子的结合情况已通过磁共振技术进行了研究。同时也对甲酸根与高铁肌红蛋白的结合进行了比较研究。高铁马肝过氧化氢酶的存在会提高甲酸根和水质子的核磁共振弛豫（NMR）速率。添加氰化物后，这种增强作用没有明显变化，这表明在氰化物存在的情况下，水和甲酸根仍然保持结合状态。通过磁共振技术测定的甲酸根质子与血红素铁的距离表明，甲酸根并不直接与过氧化氢酶或肌红蛋白的血红素铁结合，而是与球蛋白结合，并且NMR弛豫是由外层机制引起的。文中还给出了水与过氧化氢酶血红素的铁原子形成内层配合物的证据。在对高铁肌红蛋白进行的类似实验中，添加氰化物导致甲酸根和水的质子弛豫速率增强大幅下降，这分别表明水和甲酸根从血红素及其附近区域被取代。在有和没有甲酸根存在的情况下，室温下获得的过氧化氢酶和肌红蛋白的宽峰、高自旋铁离子电子顺磁共振吸收表明，甲酸根不会明显改变过氧化氢酶或肌红蛋白的血红素环境或血红素铁的自旋状态。通过可见区血红素吸收光谱变化监测甲酸根与过氧化氢酶结合的研究表明，其与血红素浓度呈一对一的化学计量关系。然而，添加甲酸根离子后在可见光谱区观察到的微小变化归因于甲酸根对血红素环境的次要影响，而非甲酸根直接与血红素部分结合。
