Unravelling the mechanisms controlling heme supply and demand

Abstract

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 (τMean) 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.

Understanding the Logistics for the Distribution of Heme in Cells

“…Free heme (either ferrous or ferric) is envisaged as being present in minuscule concentrations but will still represent a mechanism for heme to be made available in cells. 77 In (C), chaperone-mediated heme delivery to an apo -heme protein (pale red circle), for example by GAPDH. 113 − 115 In (D), heme bound to heme-binding partners (dark gray pacman) constitutes a body of exchangeable heme readily available for downstream applications, in the same way as in (B).…”

“…An ability to store heme in cells would decrease the reliance of cells on heme synthesis and degradation and could be coupled to mechanisms to make heme more or less available, on-demand and at speed ( Figure 3 D). To date, there have been no proteins identified that have the dedicated function of storing heme, but there is evidence, from recent work, 77 of a buffering capacity within cells that can accommodate changes in either the total concentration of heme (i.e., [ H t ]), or the concentration of exchangeable heme (i.e., [ H e ]), while maintain the capability to mobilize heme as and when necessary. As indicated above, it is entirely possible that known heme proteins (such as indoleamine 2,3-dioxygenase, and GAPDH) participate in the buffering of free heme concentration ([ H f ]) and protect the cell against increases or decreases in total, or exchangeable, heme concentration.…”

“…The mean fluorescence lifetime of the probe changes between the limiting values of τ apo to τ holo for the pure apo and holo forms of the sensor dependent on heme concentration. 77 (B) FRET sensors: The heme-binding domain of the sensor undergoes a conformational change that brings two fluorophores into close proximity to one another. In this example, Förster energy transfer results in a decrease in the emission of a green fluorophore and an increase in emission of a yellow fluorophore.…”

“…For a certain concentration of exchangeable heme, it is fairly straightforward to estimate an appropriate range of concentrations for the sensor that will preserve heme homeostasis in the cell. 77 On the basis of measurements in the cytosol of HEK293 cells, 77 a sensor concentration of up to 1 μM would not be expected to alter the availability of heme for exchangeable heme concentrations equal to, or in excess of, 3 μM. It is realistic to assume that, if a sensor is deployed in a localized area of the cell (e.g., directed specifically to the nucleus), then the sensor concentration may exceed 1 μM.…”

Strain engineering and bioprocessing strategies for biobased production of porphobilinogen in Escherichia coli

“…To promote PBG accumulation, we had to limit the activity of subsequent PBG-consuming reactions toward porphyrins. Since porphyrin biosynthesis is essential for cell survival, knocking out any of these PBG-consuming reactions would be lethal (Mobius et al 2010 ) (Leung et al 2021 ). Hence, we applied Clustered Regularly Interspersed Short Palindromic Repeats interference (CRISPRi) (Qi et al 2013 ) to repress the expression of hemC , whose encoding gene product of HemC mediates the conversion of PBG to HMB, with minimal impact to cell physiology.…”