Taniguchi T, Kimura T
Biochemistry. 1976 Jun 29;15(13):2849-53. doi: 10.1021/bi00658a023.
The coordination structure of the iron-sulfur center of the nitrotyrosine and the aminotyrosine derivates of bovine adrenodoxin was investigated by electron paramagnetic resonance spectroscopy. The reduced form of both modified samples exhibited signals identical with those for the native protein at g= 1.94 and g=2.01. From these results together with optical absorption and chemical analyses, it was concluded that the coordination structure of the iron-sulfur chromophore for both the derivatives was identical with the binuclear tetrahedral structure of native adrenodoxin. The configuration of the iron-binding area in nitro- and amino-adrenodoxin was studied by ovserving the circular dichroism spectra between 350 and 600 nm. The maxima for the nitro or amino derivatives were all identical with those for the native protein but different in the magnitude of their molar ellipticity. The molar ellipticities at 440 nm were 45.8 X 10(3), 14.5 X 10(3), and 9.5 X 10(6) deg cm2 per mol of iron for native adrenodoxin, nitro or amino derivative, respectively. These results suggest that the chemical modification of the tyrosine residue causes a conformational change in the iron-binding area. We have previously reported that the enzymatic activities of these reconstituted nitro and amino derivatives toware cytochrome c reduction in the presence of adrenodoxin reductase and reduced nicotinamide adenine dinucleotide phosphate were 19 and 7% of native adrenodoxin, respectively. The cytochrome c reductase activities of nitro- and aminoadrenodixin were drastically affected by the ionic strength of the assay medium, as found in native adrenodoxin. Fluorometric titration of the reductase with aminoadrenodoxin revealed that aminoadrenodoxin forms a 1:1 molar complex with the reductase. These results suggest that both the nitro and amino derivatives form a complex with the reductase. The dissociation constants of nitro- and aminoadrenodoxin for the reductase were 6.1 X 10(-7)M and 3.3 X 10(-7) M at mu = 0.04 and 1.9 X 10(-6) M and 2.0 X 10(-6) M at mu = 0.20, respectively. Comparison of these values with those of native adrenodoxin (approximately 10(-9) M at mu = 0.04 and 2.2 X 10(-7) M at mu = 0.20) suggests that an increase in the dissociation constant for the reductase is responsible for the decreased electron transferring activity of the modified adrenodoxins.
通过电子顺磁共振光谱研究了牛肾上腺皮质铁氧还蛋白的硝基酪氨酸和氨基酪氨酸衍生物中铁硫中心的配位结构。两种修饰样品的还原形式在g = 1.94和g = 2.01处呈现出与天然蛋白质相同的信号。结合这些结果以及光吸收和化学分析，得出结论：两种衍生物的铁硫发色团的配位结构与天然肾上腺皮质铁氧还蛋白的双核四面体结构相同。通过观察350至600 nm之间的圆二色光谱，研究了硝基和氨基肾上腺皮质铁氧还蛋白中铁结合区域的构型。硝基或氨基衍生物的最大值与天然蛋白质的最大值均相同，但摩尔椭圆率的大小不同。对于天然肾上腺皮质铁氧还蛋白、硝基或氨基衍生物，在440 nm处的摩尔椭圆率分别为每摩尔铁45.8×10³、14.5×10³和9.5×10⁶度厘米²/摩尔。这些结果表明，酪氨酸残基的化学修饰导致铁结合区域发生构象变化。我们之前报道过，在存在肾上腺皮质铁氧还蛋白还原酶和还原型烟酰胺腺嘌呤二核苷酸磷酸的情况下，这些重组的硝基和氨基衍生物对细胞色素c还原的酶活性分别为天然肾上腺皮质铁氧还蛋白的19%和7%。如在天然肾上腺皮质铁氧还蛋白中所发现的那样，硝基和氨基肾上腺皮质铁氧还蛋白的细胞色素c还原酶活性受到测定介质离子强度的显著影响。用氨基肾上腺皮质铁氧还蛋白对还原酶进行荧光滴定表明，氨基肾上腺皮质铁氧还蛋白与还原酶形成1:1摩尔比的复合物。这些结果表明，硝基和氨基衍生物均与还原酶形成复合物。硝基和氨基肾上腺皮质铁氧还蛋白与还原酶的解离常数在μ = 0.04时分别为6.1×10⁻⁷ M和3.3×10⁻⁷ M，在μ = 0.20时分别为1.9×10⁻⁶ M和2.0×10⁻⁶ M。将这些值与天然肾上腺皮质铁氧还蛋白的值（在μ = 0.04时约为10⁻⁹ M，在μ = 0.20时为2.2×10⁻⁷ M）进行比较表明，还原酶解离常数的增加是修饰后的肾上腺皮质铁氧还蛋白电子转移活性降低的原因。
