Heme sensor proteins

Abstract

Heme is a prosthetic group best known for roles in oxygen transport, oxidative catalysis, and respiratory electron transport. Recent years have seen the roles of heme extended to sensors of gases such as O2 and NO and cell redox state, and as mediators of cellular responses to changes in intracellular levels of these gases. The importance of heme is further evident from identification of proteins that bind heme reversibly, using it as a signal, e.g. to regulate gene expression in circadian rhythm pathways and control heme synthesis itself. In this minireview, we explore the current knowledge of the diverse roles of heme sensor proteins.

Keywords: Carbon Monoxide; Circadian Rhythms; Cytochromes; Gas Sensors; Gene Regulation; Heme; Nitric Oxide; Nuclear Receptors; Oxygen; Redox Sensor.

FIGURE 1.

Summary of key vertebrate circadian rhythm pathways. Transcription of several Period (Per) (A) and cryptochrome (Cry) (B) genes is activated by binding of the NPAS2-Bmal1 heterodimer to their 5-UTRs in forebrain or peripheral organs. In other organs, the NPAS2 paralog Clock fulfills this function. The PER and CRY proteins can also self-regulate, and their binding to the NPAS2-Bmal1 heterodimer causes dissociation from DNA and switches off their transcription (C). In addition, binding of heme and CO to NPAS2 causes dissociation of the heterodimer, switching off PER/CRY transcription (D). NPAS2-Bmal1 expression is also regulated in a heme-dependent manner (shown for NPAS2 here). In a heme-bound state, Rev-erbα recruits heterodimeric NR corepressor (NCoR) partners to repress transcription. When heme-free, it cannot repress transcription (E).

FIGURE 2.

FixL mechanism. The upper panel shows the FixL mechanism in the presence of oxygen, whereas the lower panel shows the mechanism under anaerobic conditions. FixL is shown as a red square, with the heme iron represented as a dot in the center with lines to indicate the tetrapyrrole ring. The iron is shown in purple for the high-spin ferrous state (for deoxy-FixL) and in red for the low-spin ferrous state (when bound to oxygen). In the absence of oxygen (lower panel), the His-ligated FixL dimer associates with a FixJ dimer (blue circles) to form a complex that can bind ATP. The FixL dimer can also bind ATP in the absence of FixJ but with much lower affinity. FixJ preferentially exists as a heterodimeric complex with FixL when it is non-phosphorylated. Once ATP is bound, a conserved FixL histidine residue is phosphorylated, releasing ADP. The phosphate is then transferred to a conserved aspartate residue on FixJ and stabilizes the FixJ dimer. FixJ undergoes a conformational change, and the FixL-FixJ complex dissociates. Free phosphorylated FixJ then activates transcription of target genes. Binding of O₂ (or to a lesser extent, CO or NO) causes a conformational change in FixL and switches the heme iron spin state from low spin to high spin (upper panel). CO and NO bind FixL more tightly than O₂ but are less effective inhibitors of kinase activity. In the O₂-bound state, association with FixJ and binding of ATP are unaffected, but FixL cannot be phosphorylated, and therefore FixJ cannot be switched on as a transcriptional activator. Oxygen dissociation reactivates the system (–71).

FIGURE 3.

Heme ligand binding/switching mechanisms in sGC. The heme tetrapyrrole ring is represented as a square with the ferrous (Fe²⁺) iron bound. In the resting state, the heme has His coordination with no sixth ligand (A). This form has only very low activity (red circle). Binding of CO as the sixth ligand yields a form with a 4-fold increase in activity (B; dotted green circle). At stoichiometric NO levels, distal NO binding first produces a six-coordinate species (C). The proximal His then dissociates, yielding a five-coordinate distally NO-bound form with low GC activity (D; solid green circle) (73). Alternatively, in more rapid reactions in the presence of excess NO, a second NO molecule binds at the proximal side of the heme, displacing the His (E). The distal NO then dissociates, leaving a high GC activity five-coordinate proximally NO-bound form (F; double green circle) (86). The low (D) and high (F) activity five-coordinate NO-bound forms can be distinguished by EPR spectroscopy. Preincubation of sGC substrate (Mg²⁺-GTP) or products (Mg²⁺/cGMP/PP_i) with sGC at stoichiometric NO concentrations may also lead to the high activity form (curved arrow), although ATP competes with GTP and can instead lead to formation of a low activity form (likely species D) (73). The low activity NO-bound form (D) may be less stable and more prone to deactivation in a process that may not involve dissociation of the distal NO ligand (73, 86). A distinct “desensitization” of sGC to repeated exposure to NO may result from nitrosation of a sGC protein thiol (73). The sGC allosteric stimulator YC-1 (5-(1-(phenylmethyl)-1H-indazol-3-yl)-2-furanmethanol) may convert the low activity form (D) to the high activity state (F) and also substantially stimulates the sGC activity of the CO-bound form (B). YC-1 was also reported to decrease the rate of sGC deactivation, despite enhancing the NO dissociation rate (73). Other such allosteric stimulators of sGC are also known (e.g. BAY 41-2272 (3-(4-amino-5-cyclopropylpyrimidine-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine)) (73, 85).