Regulation of Free Heme and Heme Buffering in Saccharomyces cerevisiae

Heme is an essential but potentially cytotoxic metabolite. The requirement to tightly regulate heme led to the hypothesis that intracellular free heme is managed by a cellular buffering system. Despite its importance for understanding heme trafficking and signaling, the identity and buffering capacity of this buffer remain poorly understood. In this study, we utilized the model eukaryote Saccharomyces cerevisiae and genetically encoded fluorescent heme sensors to elucidate cellular heme buffering. Towards this end, we decreased hemoprotein expression by deleting the heme-regulated transcription factor Hap1, which controls the expression of several known heme-dependent proteins, or one of its highly abundant targets, the flavohemoglobin Yhb1, and measured the effect on free heme. We found that the change in free heme and heme sensor occupancy due to ablation of Hap1 and Yhb1 is consistent with the presence of a robust cellular heme buffering system. Results from equilibrium modeling and experimental measurements of free heme are consistent with the glycolytic enzyme glyceraldehyde phosphate dehydrogenase (GAPDH) serving a role as the primary cellular heme buffer. We estimate the buffer depth to be approximately ∼50 μM and a buffering capacity {Δ[Heme]_Total / Δ[Heme]_Free} of ∼1500. As part of our studies probing the effects of Hap1 on free heme, we found that hydrogen peroxide labilizes heme, establishing a direct link between reactive oxygen species (ROS) and cellular heme availability. Finally, we show that Hap1 is required for efficient mitochondrial heme export, albeit through an unknown mechanism. Together, our studies establish fundamental metrics and regulatory mechanisms for eukaryotic heme homeostasis.