Poole L B
Department of Biochemistry, Wake Forest University Medical Center, Winston-Salem, North Carolina 27157, USA.
Biochemistry. 1996 Jan 9;35(1):65-75. doi: 10.1021/bi951888k.
The two-component alkyl hydroperoxide reductase enzyme system from Salmonella typhimurium catalyzes the pyridine nucleotide-dependent reduction of alkyl hydroperoxide and hydrogen peroxide substrates. This system is composed of a flavoenzyme, AhpF, which is related to the disulfide-reducing enzyme thioredoxin reductase, and a smaller protein, AhpC, which lacks a chromophoric cofactor. We have demonstrated that NADH-linked reduction of AhpF under anaerobic conditions converts two cystine disulfide centers to their dithiol forms. The AhpC cystine disulfide center, shown to exist as an intersubunit disulfide bond, is stoichiometrically reducible by NADH in the presence of a catalytic amount of AhpF and can be reoxidized by ethyl hydroperoxide. Disulfide bridges within oxidized AhpF form between Cys129 and Cys132 and between Cys345 and Cys348; the two C-terminal half-cystine residues, Cys476 and Cys489, exist as free thiol groups in oxidized AhpF and play no role in catalysis. Removal of the N-terminal 202-amino acid segment containing the Cys129-Cys132 disulfide center obliterates the ability of AhpF to transfer electrons to 5,5'-dthiobis(2-nitrobenzoic acid) (DTNB) and AhpC. NADH added anaerobically to AhpF causes spectral changes consistent with preferential reduction of both disulfides relative to flavin reduction; the reduction potentials of the disulfide centers are thus appropriately poised for electron transfer from NADH and flavin to disulfide-containing substrates (AhpC or DTNB), and ultimately to peroxides from AhpC. Blue, neutral flavin semiquinone is also generated in high yields during reductive titrations (91% yield during dithionite titrations), although the relatively slow formation of this species indicates its catalytic incompetence. A long wavelength absorbance band beyond 900 nm attributable to an FADH2-->NAD+ charge transfer interaction is generated during NADH, but not dithionite, titrations and may be indicative of a species directly involved in the catalytic cycle. A catalytic mechanism including the transient formation of cysteine sulfenic acid within AhpC is proposed.
鼠伤寒沙门氏菌的双组分烷基过氧化氢还原酶系统催化依赖吡啶核苷酸的烷基过氧化氢和过氧化氢底物的还原反应。该系统由一种黄素酶AhpF和一种较小的蛋白质AhpC组成，AhpF与二硫键还原酶硫氧还蛋白还原酶相关，AhpC缺乏发色辅因子。我们已经证明，在厌氧条件下，NADH连接的AhpF还原反应会将两个胱氨酸二硫键中心转化为其二硫醇形式。AhpC的胱氨酸二硫键中心以亚基间二硫键的形式存在，在催化量的AhpF存在下，能被NADH化学计量地还原，并能被乙基过氧化氢重新氧化。氧化型AhpF中的二硫键在Cys129和Cys132之间以及Cys345和Cys348之间形成；两个C末端半胱氨酸残基Cys476和Cys489在氧化型AhpF中以游离硫醇基团的形式存在，在催化过程中不起作用。去除包含Cys129 - Cys132二硫键中心的N末端202个氨基酸片段会消除AhpF将电子转移至5,5'-二硫代双(2-硝基苯甲酸)(DTNB)和AhpC的能力。厌氧条件下向AhpF中添加NADH会引起光谱变化，这与相对于黄素还原而言两个二硫键的优先还原一致；因此，二硫键中心的还原电位对于从NADH和黄素向含二硫键的底物(AhpC或DTNB)以及最终从AhpC向过氧化物的电子转移而言处于适当的状态。在还原滴定过程中也会高产率地生成蓝色的中性黄素半醌(连二亚硫酸钠滴定过程中的产率为91%)，尽管该物种形成相对较慢表明其无催化活性。在NADH滴定而非连二亚硫酸钠滴定过程中会产生一个超过900 nm的长波长吸收带，这归因于FADH2→NAD+电荷转移相互作用，并且可能表明存在一种直接参与催化循环的物种。本文提出了一种催化机制，其中包括AhpC内半胱氨酸亚磺酸的瞬时形成。
