Kostic Milka, Bernhardt Rita, Pochapsky Thomas C
Department of Chemistry, Brandeis University, 415 South Street, MS 015, Waltham, Massachusetts 02451, USA.
Biochemistry. 2003 Jul 15;42(27):8171-82. doi: 10.1021/bi034500r.
Adrenodoxin (Adx) belongs to the family of Cys(4)Fe(2)S(2) vertebrate-type ferredoxins that shuttle electrons from NAD(P)H-dependent reductases to cytochrome P450 enzymes. The vertebrate-type ferredoxins contain a conserved basic residue, usually a histidine, adjacent to the third cysteine ligand of the Cys(4)Fe(2)S(2) cluster. In bovine Adx the side chain of this residue, His 56, is involved in a hydrogen-bonding network within the domain of Adx that interacts with redox partners. It has been proposed that this network acts as a mechanical link between the metal cluster binding site and the interaction domain, transmitting redox-dependent conformational or dynamical changes from the cluster binding loop to the interaction domain. H/D exchange studies indicate that oxidized Adx (Adx(o)) is more dynamic than reduced Adx (Adx(r)) on the kilosecond time scale in many regions of the protein, including the interaction domain. Dynamical differences on picosecond to nanosecond time scales between the oxidized (Adx(o)) and reduced (Adx(r)) adrenodoxin were probed by measurement of (15)N relaxation parameters. Significant differences between (15)N R(2) rates were observed for all residues that could be measured, with those rates being faster in Adx(o) than in Adx(r). Two mutations of His 56, H56R and H56Q, were also characterized. No systematic redox-dependent differences between (15)N R(2) rates or H/D exchange rates were observed in either mutant, indicating that His 56 is required for the redox-dependent behavior observed in WT Adx. Comparison of chemical shift differences between oxidized and reduced H56Q and H56R Adx confirms that redox-dependent changes are smaller in these mutants than in the wild-type Adx.
肾上腺皮质铁氧化还原蛋白(Adx)属于Cys(4)Fe(2)S(2)脊椎动物型铁氧化还原蛋白家族,它将电子从依赖NAD(P)H的还原酶传递给细胞色素P450酶。脊椎动物型铁氧化还原蛋白在Cys(4)Fe(2)S(2)簇的第三个半胱氨酸配体附近含有一个保守的碱性残基,通常是组氨酸。在牛Adx中,这个残基(His 56)的侧链参与了Adx结构域内与氧化还原伙伴相互作用的氢键网络。有人提出,这个网络作为金属簇结合位点和相互作用结构域之间的机械连接,将氧化还原依赖性的构象或动力学变化从簇结合环传递到相互作用结构域。氢/氘交换研究表明,在蛋白质的许多区域,包括相互作用结构域,氧化型Adx(Adx(o))在千秒时间尺度上比还原型Adx(Adx(r))更具动态性。通过测量(15)N弛豫参数,探究了氧化型(Adx(o))和还原型(Adx(r))肾上腺皮质铁氧化还原蛋白在皮秒到纳秒时间尺度上的动力学差异。对于所有可测量的残基,观察到(15)N R(2)速率存在显著差异,Adx(o)中的这些速率比Adx(r)中的更快。还对His 56的两个突变体H56R和H56Q进行了表征。在这两个突变体中均未观察到(15)N R(2)速率或氢/氘交换速率之间存在系统的氧化还原依赖性差异,这表明His 56是野生型Adx中观察到的氧化还原依赖性行为所必需的。氧化型和还原型H56Q和H56R Adx之间化学位移差异的比较证实,这些突变体中氧化还原依赖性变化比野生型Adx中的小。