Lode E T, Murray C L, Rabinowitz J C
J Biol Chem. 1976 Mar 25;251(6):1683-7.
The effects of pH and ionic strength on the midpoint reduction potential (Emp) of Clostridium acidi-urici ferredoxin were determined using hydrogen gas and hydrogenase. The Emp of native ferredoxin at 24-25 degrees in 0.1 M Tris-chloride buffer, pH 7.0, is--0.434 V. In the pH range examined, the Emp becomes approximately 13 mv more negative per each pH unit increase. A plot of the log of ionic strength versus the apparent Emp of ferredoxin in 0.1 M Tris-chloride buffer, pH 7.5, Was linear over the range of 1.0 to 0.01 ionic strength with Emp values of--0.414 and--0.475 V, respectively, at these extremes. This effect is the same with sodium chloride, sodium bromide, or ammonium sulfate. Potassium phosphate buffer caused a similar change, but the absolute values of Emp differed from those obtained in the presence of the other salts. This effect of pH and ionic strength on Emp may be general for clostridial-type (Fe4S4)2-ferredoxins, since the apparent Emp of Clostridium pasteurianum ferredoxin is affected in a similar manner by these two variables. The Emp of this ferredoxin in 0.1 M Tris-chloride buffer pH 7.0, is--0.405 V. Since the NH2-terminal amino acid residue, Ala1, and Tyr2 of C. acidi urici ferredoxin are near an (Fe4S4)2-cluster in the protein, the apparent Emp of derivatives that contained amino acid replacements in these two positions were determined. Under similar conditions, the Emp of most of the 13 derivatives examined, including those of [Leu2]- and[3-NH2-Tyr30]ferredoxin, is approximately the same as that of native ferredoxin. However, the Emp of [His2]ferredoxin is approximately 15 mv more positive, whereas that of [Trp2]ferredoxin is 22 mv more negative than that of native C. acidi-urici ferredoxin. Variations in sodium chloride concentration and pH also affected the apparent Emp of the derivatives. It is suggested that the changes observed in the Emp of C. acidi-urici ferredoxin are caused by protein conformational changes.
使用氢气和氢化酶测定了pH值和离子强度对尿酸梭菌铁氧化还原蛋白中点还原电位（Emp）的影响。在0.1M三氯化物缓冲液（pH 7.0）中，24 - 25℃下天然铁氧化还原蛋白的Emp为 - 0.434V。在所研究的pH范围内，每增加一个pH单位，Emp大约变得更负13mV。在0.1M三氯化物缓冲液（pH 7.5）中，离子强度的对数与铁氧化还原蛋白的表观Emp的关系图在离子强度1.0至0.01的范围内呈线性，在这些极端情况下，Emp值分别为 - 0.414V和 - 0.475V。氯化钠、溴化钠或硫酸铵的情况相同。磷酸钾缓冲液引起类似变化，但Emp的绝对值与其他盐存在时获得的值不同。pH值和离子强度对Emp的这种影响可能对梭菌型（Fe4S4）2 - 铁氧化还原蛋白是普遍的，因为巴氏梭菌铁氧化还原蛋白的表观Emp受这两个变量的影响方式类似。在0.1M三氯化物缓冲液（pH 7.0）中，这种铁氧化还原蛋白的Emp为 - 0.405V。由于尿酸梭菌铁氧化还原蛋白的NH2 - 末端氨基酸残基Ala1和Tyr2靠近蛋白质中的（Fe4S4）2 - 簇，因此测定了在这两个位置含有氨基酸替换的衍生物的表观Emp。在类似条件下，所研究的13种衍生物中的大多数，包括[Leu2] - 和[3 - NH2 - Tyr30]铁氧化还原蛋白的衍生物，其Emp与天然铁氧化还原蛋白的Emp大致相同。然而，[His2]铁氧化还原蛋白的Emp大约更正15mV，而[Trp2]铁氧化还原蛋白的Emp比天然尿酸梭菌铁氧化还原蛋白的Emp更负22mV。氯化钠浓度和pH值的变化也影响了衍生物的表观Emp。有人认为，尿酸梭菌铁氧化还原蛋白Emp中观察到的变化是由蛋白质构象变化引起的。
