Abstract：
Nitric oxide (˙NO) is a free radical that induces nitrosative stress, which can jeopardize cell viability. Yeasts have evolved diverse detoxification mechanisms to effectively counteract ˙NO-mediated cytotoxicity. One mechanism relies on the flavohemoglobin Yhb1, whereas a second one requires the S-nitrosoglutathione reductase Fmd2. To investigate heme-dependent activation of Yhb1 in response to ˙NO, we use hem1Δ-derivative Schizosaccharomyces pombe strains lacking the initial enzyme in heme biosynthesis, forcing cells to assimilate heme from external sources. Under these conditions, yhb1+ mRNA levels are repressed in the presence of iron through a mechanism involving the GATA-type transcriptional repressor Fep1. In contrast, when iron levels are low, the transcription of yhb1+ is derepressed and further induced in the presence of the ˙NO donor DETANONOate. Cells lacking Yhb1 or expressing inactive forms of Yhb1 fail to grow in a hemin-dependent manner when exposed to DETANONOate. Similarly, the loss of function of the heme transporter Str3 phenocopies the effects of Yhb1 disruption by causing hypersensitivity to DETANONOate under hemin-dependent culture conditions. Coimmunoprecipitation and bimolecular fluorescence complementation assays demonstrate the interaction between Yhb1 and the heme transporter Str3. Collectively, our findings unveil a novel pathway for activating Yhb1, fortifying yeast cells against nitrosative stress.
摘要：
Nitricoxide(•NO)是一种诱导亚硝基应激的自由基,这会危及细胞活力。酵母菌已经进化出多种解毒机制，以有效地抵消*NO介导的细胞毒性。一种机制依赖于黄素血红蛋白Yhb1，而第二种机制需要S-亚硝基谷胱甘肽还原酶Fmd2。为了研究血红素依赖性激活Yhb1以响应苄NO，我们使用缺乏血红素生物合成初始酶的hem1Δ衍生裂殖酵母菌株，迫使细胞从外部来源吸收血红素。在这些条件下,在铁的存在下，通过涉及GATA型转录阻遏物Fep1的机制来抑制yhb1mRNA水平。相比之下,当铁含量低时，yhb1的转录被抑制，并在有*NO供体DETANOate的情况下进一步诱导。缺乏Yhb1或表达Yhb1非活性形式的细胞在暴露于DETanonoate时不能以血红素依赖性方式生长。同样，血红素转运蛋白Str3功能的丧失通过在血红素依赖性培养条件下引起对DETANONA酸盐的超敏反应来表现Yhb1破坏的作用。共免疫沉淀和双分子荧光互补测定证明了Yhb1和血红素转运蛋白Str3之间的相互作用。总的来说，我们的发现揭示了激活Yhb1的新途径，强化酵母细胞对抗亚硝化应激。
