Bonomi Francesco, Eidsness Marly K, Iametti Stefania, Kurtz Donald M, Mazzini Stefania, Morleo Anna
DISMA, Via Celoria 2, 20133, Milan, Italy.
J Biol Inorg Chem. 2004 Apr;9(3):297-306. doi: 10.1007/s00775-004-0525-4. Epub 2004 Feb 10.
The thermostabilities of Fe(2+) ligation in rubredoxins (Rds) from the hyperthermophile Pyrococcus furiosus (Pf) and the mesophiles Clostridium pasteurianum (Cp) and Desulfovibrio vulgaris (Dv) were compared. Residue 44 forms an NH.S(Cys) hydrogen bond to one of the cysteine ligands to the [Fe(SCys)(4)] site, and substitutions at this location affect the redox properties of the [Fe(SCys)(4)] site. Both Pf Rd and Dv Rd have an alanine residue at position 44, whereas Cp Fd has a valine residue. Wild-type proteins were examined along with V44A and A44V "exchange" mutants of Cp and Pf Rds, respectively, in order to assess the effects of the residue at position 44 on the stability of the [Fe(SCys)(4)] site. Stability of iron ligation was measured by temperature-ramp and fixed-temperature time course experiments, monitoring iron release in both the absence and presence of more thiophilic metals (Zn(2+), Cd(2+)) and over a range of pH values. The thermostability of the polypeptide fold was concomitantly measured by fluorescence, circular dichroism, and (1)H NMR spectroscopies. The A44V mutation strongly lowered the stability of the [Fe(II)(SCys)(4)] site in Pf Rd, whereas the converse V44A mutation of Cp Rd significantly raised the stability of the [Fe(II)(SCys)(4)] site, but not to the levels measured for wild-type Dv Rd. The region around residue 44 is thus a significant contributor to stability of iron coordination in reduced Rds. This region, however, made only a minor contribution to the thermostability of the protein folding, which was found to be higher for hyperthermophilic versus mesophilic Rds, and largely independent of the residue at position 44. These results, together with our previous studies, show that localized charge density, solvent accessibility, and iron site/backbone interactions control the thermostability of the [Fe(SCys)(4)] site. The iron site thermostability does make a minor contribution to the overall Rd thermostability. From a mechanistic standpoint, we also found that attack of displacing ions (H(+), Cd(2+)) on the Cys42 sulfur ligand at the [Fe(SCys)(4)] site occurs through the V8 side and not the V44 side of the iron site.
对嗜热栖热菌（Pf）、嗜温菌巴氏芽孢梭菌（Cp）和脱硫弧菌（Dv）的红素氧还蛋白（Rds）中Fe(2+)配体的热稳定性进行了比较。44位残基与[Fe(SCys)(4)]位点的一个半胱氨酸配体形成NH.S(Cys)氢键，该位置的取代会影响[Fe(SCys)(4)]位点的氧化还原性质。Pf Rd和Dv Rd在44位均有一个丙氨酸残基，而Cp Fd在该位置有一个缬氨酸残基。分别对Cp和Pf Rds的野生型蛋白以及V44A和A44V“交换”突变体进行了检测，以评估44位残基对[Fe(SCys)(4)]位点稳定性的影响。通过温度梯度和恒温时间进程实验来测量铁配体的稳定性，监测在有无更多亲硫金属（Zn(2+)、Cd(2+)）存在的情况下以及在一系列pH值范围内的铁释放情况。通过荧光、圆二色性和(1)H NMR光谱同时测量多肽折叠的热稳定性。A44V突变显著降低了Pf Rd中[Fe(II)(SCys)(4)]位点的稳定性，而Cp Rd相反的V44A突变则显著提高了[Fe(II)(SCys)(4)]位点的稳定性，但未达到野生型Dv Rd所测的水平。因此，44位残基周围区域对还原态Rds中铁配位的稳定性有重要贡献。然而，该区域对蛋白质折叠的热稳定性贡献较小，嗜热Rds的热稳定性高于嗜温Rds，且在很大程度上与44位残基无关。这些结果与我们之前的研究一起表明，局部电荷密度、溶剂可及性以及铁位点/主链相互作用控制着[Fe(SCys)(4)]位点的热稳定性。铁位点的热稳定性对整体Rd热稳定性确实有较小贡献。从机理角度来看，我们还发现，在[Fe(SCys)(4)]位点，取代离子（H(+)、Cd(2+)）对Cys42硫配体的攻击是通过铁位点的V8侧而非V44侧发生的。
