Adzamli I K, Petrou A, Sykes A G, Rao K K, Hall D O
Biochem J. 1983 Apr 1;211(1):219-26. doi: 10.1042/bj2110219.
Kinetic results are presented for the reaction of reduced [2Fe-2S] ferredoxin from the blue-green alga Spirulina platensis with Co(NH3)6(3+), Co(edta)- and Co(acac)3 as oxidants at pH 8.0 at I0.10 (NaCl). The aim is to compare results obtained with those previously reported for the [2Fe-2S] ferredoxin from parsley, where the two ferredoxins under consideration are in evolutionary terms widely divergent (35% amino acid variations). The three oxidants chosen have different ligand sets and different charges, and are the complexes that in previous studies have given greatest diversity in behaviour. With Co(NH3)6(3+) first-order rate constants (oxidant in large excess) tend to a limiting value with increasing concentration of oxidant. With Co(edta)- and Co(acac)3 there is no similar tendency to limiting behaviour and a first-order dependence on oxidant is observed. The temperature-dependence of the Co(NH3)6(3+) reaction was investigated, and values were obtained for delta H0 [19.8kJ X mol-1 (4.7kcal X mol-1)] and delta S0 [129.3J X K-1 X mol-1 (30.9 cal X K-1 X mol-1)] for the association step that occurs before electron transfer. Whereas redox-inactive Cr(NH3)6(3+) displays competitive inhibition in the reaction of Co(NH3)6(3+), it accelerates the reaction of Co(edta)-, and only partially blocks the reaction with Co(acac)3. Results obtained are similar to those previously reported for parsley (and spinach) ferredoxin. It is concluded that electrostatics play a dominant role and that a negatively charged functional site on the protein common to all three ferredoxins is influential. Conserved negative patches at positions 67-69 and 94-96 within 1.0 nm (10A) of an Fe atom of the active site, as well as the exposed S atoms of cysteine residues 41 and 46, which are a part of the Fe2S*2(SR)4(3-) cluster, are the most likely possibilities. The various effects of Cr(NH3)6(3+) provide a means of testing for utilization of the same site in reactions of the ferredoxins with physiological partners.
给出了来自蓝藻钝顶螺旋藻的还原型[2Fe-2S]铁氧化还原蛋白与作为氧化剂的Co(NH₃)₆³⁺、Co(edta)⁻和Co(acac)₃在pH 8.0、I 0.10（NaCl）条件下反应的动力学结果。目的是将所得结果与先前报道的来自欧芹的[2Fe-2S]铁氧化还原蛋白的结果进行比较，所考虑的这两种铁氧化还原蛋白在进化上差异很大（氨基酸变异35%）。所选的三种氧化剂具有不同的配体组和不同的电荷，并且是在先前研究中行为表现出最大多样性的配合物。对于Co(NH₃)₆³⁺，一级速率常数（氧化剂大大过量）随着氧化剂浓度的增加趋向于一个极限值。对于Co(edta)⁻和Co(acac)₃，没有类似的趋向于极限行为的趋势，并且观察到对氧化剂的一级依赖性。研究了Co(NH₃)₆³⁺反应的温度依赖性，并获得了电子转移前发生的缔合步骤的ΔH⁰[19.8kJ·mol⁻¹（4.7kcal·mol⁻¹）]和ΔS⁰[129.3J·K⁻¹·mol⁻¹（30.9cal·K⁻¹·mol⁻¹）]值。虽然氧化还原惰性的Cr(NH₃)₆³⁺在Co(NH₃)₆³⁺的反应中表现出竞争性抑制，但它加速了Co(edta)⁻的反应，并且仅部分阻断了与Co(acac)₃的反应。所得结果与先前报道的欧芹（和菠菜）铁氧化还原蛋白的结果相似。得出的结论是，静电作用起主导作用，并且所有三种铁氧化还原蛋白共有的蛋白质上带负电荷的功能位点具有影响力。活性位点的一个Fe原子的1.0nm（10Å）范围内67 - 69位和94 - 96位的保守负性区域，以及作为Fe₂S₂(SR)₄³⁻簇一部分的半胱氨酸残基41和46的暴露S原子，是最有可能的情况。Cr(NH₃)₆³⁺的各种效应提供了一种测试铁氧化还原蛋白与生理伙伴反应中是否利用同一位点的方法。
