Irreversible photoreduction of flavin in a mutated Phot-LOV1 domain
Irreversible photoreduction of flavin in a mutated Phot-LOV1 domain
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DOI：
10.1021/bi034863r
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发表时间：
2003-08-26
期刊：
影响因子：
2.9
通讯作者：
Hegemann, P
中科院分区：
文献类型：
--
作者：
Kottke, T;Dick, B;Hegemann, P
热感受器是一个重要的蛋白质家族，调节蓝光反应的生物过程。它们包含两个光、氧和电压敏感(LOV)结构域和一个丝氨酸/苏氨酸激酶结构域。两个LOV结构域都非共价结合一个黄素单核苷酸(FMN)。在蓝光吸收后，LOV结构域经历一个光循环，瞬间形成半胱氨酸残基和FMN的共价加合物(LOV-390)。黄素-硫醇加合物的形成机制尚不清楚。我们研究了莱茵衣藻LOV1结构域的一个突变体，用蛋氨酸取代了反应性半胱氨酸57(C57M)。与野生型一样，辐射会导致光加合物的形成，但它会不可逆转地转化为红色吸收物种C57M675。根据光谱结果和2.1(A)的超帽分辨晶体结构，这种极不寻常的FMN物种被归属于N(5)位上与脱辅基蛋白共价连接的中性黄素自由基。与其他黄素蛋白中性自由基相比，C57M-675即使在有氧或变性条件下也是稳定的。对于C57M突变体和野生型LOV1结构域，讨论了光诱导形成加合物的途径。
Phot photoreceptors make up an important protein family regulating biological processes in response to blue light. They contain two light, oxygen, and voltage sensitive (LOV) domains and a serine/threonine kinase domain. Both LOV domains noncovalently bind a flavin mononucleotide (FMN). Upon absorption of blue light, the LOV domains undergo a photocycle, transiently forming a covalent adduct of a cysteine residue and the FMN (LOV-390). The mechanism of formation of this flavin-thiol adduct is still unclear. We studied a mutant of the LOV1 domain from the green alga Chlamydomonas reinhardtii with a methionine replacing the reactive cysteine 57 (C57M). As in the wild type, irradiation leads to formation of a photoadduct, which, however, is irreversibly converted into a red absorbing species, C57M675. On the basis of spectroscopic results and the 2.1 (A) over cap resolution crystal structure, this highly unusual FMN species was assigned to a neutral flavin radical covalently attached to the apoprotein at the N(5) position. In contrast to other flavoprotein neutral radicals, C57M-675 is stable even under aerobic or denaturing conditions. Pathways for the photoinduced formation of the adduct are discussed for the C57M mutant as well as the wild-type LOV1 domain.