Iron protoporphyrin IX (heme) is a redox-active cofactor that is bound in mammalian cells by GAPDH and allocated by a process influenced by physiologic levels of NO. This impacts the activity of many heme proteins including indoleamine dioxygenase-1 (IDO1), a redox enzyme involved in immune response and tumor growth. To gain further understanding we created a tetra-Cys human GAPDH reporter construct (TC-hGAPDH) which after labeling could indicate its heme binding by fluorescence quenching. When purified or expressed in a human cell line, TC-hGAPDH had properties like native GAPDH and heme binding quenched its fluorescence by 45–65%, allowing it to report on GAPDH binding of mitochondrially-generated heme in live cells in real time. In cells with active mitochondrial heme synthesis, low-level NO exposure increased heme allocation to IDO1 while keeping the TC-hGAPDH heme level constant due to replenishment by mitochondria. When mitochondrial heme synthesis was blocked, low NO caused a near complete transfer of the existing heme in TC-hGAPDH to IDO1 in a process that required IDO1 be able to bind the heme and have an active hsp90 present. Higher NO exposure had the opposite effect and caused IDO1 heme to transfer back to TC-hGAPDH. This demonstrated: (i) flow of mitochondrial heme through GAPDH is tightly coupled to target delivery, (ii) NO up- or down-regulates IDO1 activity by promoting a conserved heme exchange with GAPDH that goes in either direction according to the NO exposure level. The ability to drive a concentration-dependent, reversible protein heme exchange is unprecedented and reveals a new role for NO in biology.

中文翻译：

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可视化活细胞中通过 GAPDH 的线粒体血红素流动及其受 NO 的调节


铁原卟啉 IX（血红素）是一种氧化还原活性辅助因子，在哺乳动物细胞中通过 GAPDH 结合，并通过受 NO 生理水平影响的过程进行分配。这会影响许多血红素蛋白的活性，包括吲哚胺双加氧酶-1 (IDO1)，这是一种参与免疫反应和肿瘤生长的氧化还原酶。为了进一步了解，我们创建了四半胱氨酸人 GAPDH 报告构建体 (TC-hGAPDH)，标记后可以通过荧光猝灭表明其血红素结合。当在人类细胞系中纯化或表达时，TC-hGAPDH 具有类似于天然 GAPDH 的特性，并且血红素结合将其荧光猝灭 45-65%，使其能够实时报告活细胞中线粒体生成的血红素的 GAPDH 结合。在线粒体血红素合成活跃的细胞中，低水平的 NO 暴露增加了血红素向 IDO1 的分配，同时由于线粒体的补充而保持 TC-hGAPDH 血红素水平恒定。当线粒体血红素合成受阻时，低 NO 导致 TC-hGAPDH 中现有血红素几乎完全转移到 IDO1，该过程需要 IDO1 能够结合血红素并存在活性 hsp90。较高的 NO 暴露会产生相反的效果，导致 IDO1 血红素转移回 TC-hGAPDH。这证明：(i) 线粒体血红素通过 GAPDH 的流动与目标递送紧密耦合，(ii) NO 通过促进与 GAPDH 的保守血红素交换来上调或下调 IDO1 活性，该交换根据 NO 暴露水平向任一方向进行。驱动浓度依赖性、可逆的蛋白质血红素交换的能力是前所未有的，揭示了 NO 在生物学中的新作用。