A conserved histidine in vertebrate-type ferredoxins is critical for redox-depen

2019-07-14 12:25:12

rates domain dependent redox Adx

责任者: Kostic, Milka;Bernhardt, Rita;Pochapsky, Thomas C. 单位: Department of Chemistry, Brandeis University, MS 015, Waltham, MA 02451, United States 来源出处: Biochemistry,2003,42(27):8171-8182 摘要: Adrenodoxin (Adx) belongs to the family of Cys4Fe2S2 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 Cys4Fe2S2 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°) is more dynamic than reduced Adx (Adxr) 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°) and reduced (Adxr) adrenodoxin were probed by measurement of 15N relaxation parameters. Significant differences between 15N R2 rates were observed for all residues that could be measured, with those rates being faster in Adx° than in Adxr. Two mutations of His 56, H56R and H56Q, were also characterized. No systematic redox-dependent differences between 15N R2 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. 关键词: Iron compounds;Enzyme kinetics;Hydrogen bonds;Oxidation;Reduction;Redox reactions;Ligands