Indoleamine Dioxygenase and Tryptophan Dioxygenase Activities are Regulated through Control of Cell Heme Allocation by Nitric Oxide

Abstract

Indoleamine-2, 3-dioxygenase (IDO1) and Tryptophan-2, 3-dioxygenase (TDO) catalyze the conversion of L-tryptophan to N-formyl- kynurenine and thus play primary roles in metabolism, inflammation, and tumor immune surveillance. Because their activities depend on their heme contents which range from 30- 60% heme-saturated in biological settings and go up or down in a dynamic manner, we studied how their heme levels may be impacted by nitric oxide (NO) in mammalian cells. We utilized cells expressing TDO or IDO1 either naturally or via transfection and determined their activities, heme contents, and expression levels as a function of NO exposure. We found NO has a bimodal effect: A narrow range of very low NO exposure promoted cells to allocate heme into TDO and IDO1 and boosted their activities several fold, while beyond this range the NO exposure transitioned to have a negative impact on their heme contents and activities. NO did not alter dioxygenase protein expression levels and its bimodal impact was observed when NO was released by a chemical donor or was generated naturally by immune-stimulated macrophage cells. NO-driven heme allocations to IDO1 and TDO required participation of a GAPDH- heme complex and for IDO1 required chaperone Hsp90 activity. Thus, cells can up- or down-regulate their IDO1 and TDO activities through a bimodal control of heme allocation by NO. This mechanism has important biomedical implications and helps explain why the IDO1 and TDO activities in animals go up and down in response to immune stimulation.

A natural heme deficiency exists in biology that allows nitric oxide to control heme protein functions by regulating cellular heme distribution

“…In all cases examined so far, there is a relatively narrow window of very low NO concentration that stimulates cell heme allocation, and as the NO concentration goes above this range it increasingly has a negative impact that ultimately overcomes the positive effect and even can lead to a net loss of heme from the heme proteins. [ 8,22,31,37 ] In all cases tested, the NO‐driven heme allocations require the participation of GAPDH and Hsp90, indicating that the NO‐driven mechanism involves the same cellular machinery as does normal heme allocation. One mechanism (among several) for NO‐driven cell heme allocation that is consistent with the results to date is shown in Figure 2, using sGC as an example heme protein.…”

“…The easiest and oldest method is to measure the heme‐dependent functional activity of a given heme protein of interest in a biological sample in the absence or presence of added heme. This has been used for example to indicate the percentage heme saturation in TDO, IDO‐1, sGC, and cytochromes P450 in homogenates of various cells or tissues, [ 8,14‐16,59,60 ] but its usefulness depends on the ability of the given heme protein of interest to be amenable to in vitro heme reconstitution. Another way would be to determine the heme levels in the heme proteins by mass spectrometry.…”

“…[ 39 ] The different impacts of the NO synthase isoforms on cell heme allocation as described above have been borne out in cell culture experiments. [ 8,22 ]…”

Visualizing Mitochondrial Heme Flow through GAPDH to Targets in Living Cells and its Regulation by NO

“…Specifically, relatively low level NO exposure promoted cell heme allocation and thus caused the heme content of several heme proteins to increase by a process that also depended on GAPDH and Hsp90. In contrast, at higher NO exposure levels the promoting effect of NO was quickly lost and instead it widely prevented cellular heme allocation to proteins (17,18) and even caused some to lose their heme (17). NO displayed this hormetic effect when it was released from small molecule NO donors or when it was naturally generated in cells via NO synthases (17,19-21).…”

“…In contrast, at higher NO exposure levels the promoting effect of NO was quickly lost and instead it widely prevented cellular heme allocation to proteins (17,18) and even caused some to lose their heme (17). NO displayed this hormetic effect when it was released from small molecule NO donors or when it was naturally generated in cells via NO synthases (17,19-21). Overall, this suggests a new way that NO can shape biological processes by positively or negatively regulating cell heme allocation and the consequent functions of numerous heme-dependent proteins.…”