Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida , Orlando, Florida 32827, United States.
Biochemistry. 2014 Mar 4;53(8):1342-9. doi: 10.1021/bi5000349. Epub 2014 Feb 19.
The diheme enzyme MauG catalyzes a six-electron oxidation that is required for the posttranslational modification of a precursor of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived cofactor, tryptophan tryptophylquinone (TTQ). Crystallographic and computational studies have implicated Gln103 in stabilizing the Fe(IV)═O moiety of the bis-Fe(IV) state by hydrogen bonding. The role of Gln103 was probed by site-directed mutagenesis. Q103L and Q103E mutations resulted in no expression and very little expression of the protein, respectively. Q103A MauG exhibited oxidative damage when isolated. Q103N MauG was isolated at levels comparable to that of wild-type MauG and exhibited normal activity in catalyzing the biosynthesis of TTQ from preMADH. The crystal structure of the Q103N MauG-preMADH complex suggests that a water may mediate hydrogen bonding between the shorter Asn103 side chain and the Fe(IV)═O moiety. The Q103N mutation caused the two redox potentials associated with the diferric/diferrous redox couple to become less negative, although the redox cooperativity of the hemes of MauG was retained. Upon addition of H2O2, Q103N MauG exhibits changes in the absorbance spectrum in the Soret and near-IR regions consistent with formation of the bis-Fe(IV) redox state. However, the rate of spontaneous return of the spectrum in the Soret region was 4.5-fold greater for Q103N MauG than for wild-type MauG. In contrast, the rate of spontaneous decay of the absorbance at 950 nm, which is associated with charge-resonance stabilization of the high-valence state, was similar for wild-type MauG and Q103N MauG. This suggests that as a consequence of the mutation a different distribution of resonance structures stabilizes the bis-Fe(IV) state. These results demonstrate that subtle changes in the structure of the side chain of residue 103 can significantly affect the overall protein stability of MauG and alter the redox properties of the hemes.
二血红素酶 MauG 催化六电子氧化，这是甲基胺脱氢酶 (preMADH) 前体翻译后修饰所必需的，以完成其蛋白衍生辅因子色氨酸色氨酸醌 (TTQ) 的生物合成。晶体学和计算研究表明 Gln103 通过氢键稳定双 Fe(IV)状态的 Fe(IV)═O 部分。通过定点突变研究了 Gln103 的作用。Q103L 和 Q103E 突变分别导致蛋白无表达和很少表达。分离时，Q103A MauG 表现出氧化损伤。Q103N MauG 分离水平与野生型 MauG 相当，并表现出正常催化 preMADH 生成 TTQ 的活性。Q103N MauG-preMADH 复合物的晶体结构表明，一个水分子可能介导较短的 Asn103 侧链与 Fe(IV)═O 部分之间的氢键。Q103N 突变导致与双铁/亚铁氧化还原对相关的两个氧化还原电位变得不那么负，尽管 MauG 的血红素的氧化还原协同性得以保留。加入 H2O2 后，Q103N MauG 在 Soret 和近红外区域的吸收光谱中表现出与双 Fe(IV)氧化还原态形成一致的变化。然而，Q103N MauG 在 Soret 区域自发恢复光谱的速率比野生型 MauG 快 4.5 倍。相比之下，与高价态电荷共振稳定相关的 950nm 处吸收的自发衰减速率在野生型 MauG 和 Q103N MauG 之间相似。这表明，由于突变，不同的共振结构分布稳定了双 Fe(IV)状态。这些结果表明，残基 103 侧链结构的细微变化会显著影响 MauG 的整体蛋白稳定性，并改变血红素的氧化还原性质。
