Unravelling the mechanisms controlling heme supply and demand

In addition to heme’s role as the prosthetic group buried inside many different proteins that are ubiquitous in biology, there is new evidence that heme has substantive roles in cellular signaling and regulation. This means that heme must be available in locations distant from its place of synthesis (mitochondria) in response to transient cellular demands. A longstanding question has been to establish the mechanisms that control the supply and demand for cellular heme. By fusing a monomeric heme-binding peroxidase (ascorbate peroxidase, mAPX) to a monomeric form of green-fluorescent protein (mEGFP), we have developed a heme sensor (mAPXmEGFP) that can respond to heme availability. By means of fluorescence lifetime imaging, this heme sensor can be used to quantify heme concentrations; values of the mean fluorescence lifetime (τ(Mean)) for mAPX-mEGFP are shown to be responsive to changes in free (unbound) heme concentration in cells. The results demonstrate that concentrations are typically limited to one molecule or less within cellular compartments. These miniscule amounts of free heme are consistent with a system that sequesters the heme and is able to buffer changes in heme availability while retaining the capability to mobilize heme when and where it is needed. We propose that this exchangeable supply of heme can operate using mechanisms for heme transfer that are analogous to classical ligand-exchange mechanisms. This exquisite control, in which heme is made available for transfer one molecule at a time, protects the cell against the toxic effect of excess heme and offers a simple mechanism for heme-dependent regulation in single-molecule steps.