Haem is essential for living organisms, functioning as a crucial element in the redox-sensitive reaction centre in haemproteins. During the biogenesis of these proteins, the haem cofactor is typically incorporated enzymatically into the haem pockets of the apo-haemprotein as the functionally indispensable prosthetic group. A class of ion channel, the large-conductance calcium-dependent Slo1 BK channels, possesses a conserved haem-binding sequence motif. Here we present electrophysiological and structural evidence showing that haem directly regulates cloned human Slo1 channels and wild-type BK channels in rat brain. Both oxidized and reduced haem binds to the hSlo1 channel protein and profoundly inhibits transmembrane K+ currents by decreasing the frequency of channel opening. This direct regulation of the BK channel identifies a previously unknown role of haem as an acute signalling molecule.

The CO-sensing mechanism of the transcription factor CooA from Rhodospirillum rubrum was studied through a systematic mutational analysis of potential heme ligands. Previous electron paramagnetic resonance (EPR) spectroscopic studies on wild-type CooA suggested that oxidized (FeIII) CooA contains a low-spin heme with a thiolate ligand, presumably a cysteine, bound to its heme iron. In the present report, electronic absorption and EPR analysis of various substitutions at Cys residues establish that Cys75 is a heme ligand in FeIII CooA. However, characterization of heme stability and electronic properties of purified C75S CooA suggest that Cys75 is not a ligand in FeII CooA. Mutational analysis of all CooA His residues showed that His77 is critical for CO-stimulated transcription. On the basis of findings that H77Y CooA is perturbed in its FeII electronic properties and is unable to bind DNA in a site-specific manner in response to CO, His77 appears to be an axial ligand to FeII CooA. These results imply a ligand switch from Cys75 to His77 upon reduction of CooA. In addition, an interaction has been identified between Cys75 and His77 in FeIII CooA that may be involved in the CO-sensing mechanism. Finally, His77 is necessary for the proper conformational change of CooA upon CO binding.

Electronic absorption, EPR, and resonance Raman spectroscopies revealed that CooA, the CO-sensing transcriptional regulator from Rhodospirillum rubrum, reacts with NO to form a five-coordinate NO-heme. NO must therefore displace both of the heme ligands from six-coordinate, low-spin Fe(II)CooA in forming five-coordinate Fe(II)CooA(NO). CO, in contrast, displaces a single heme ligand from Fe(II)CooA to form six-coordinate Fe(II)CooA(CO). Of a series of common heme-binding ligands, only CO and NO were able to bind to the heme of wild-type CooA; imidazole, azide anion, and cyanide anion had no effect on the heme absorption spectrum. Although NO binds to the heme and displaces the endogenous ligands, NO was not able to induce CooA to bind to its target DNA. The mechanism of CO-dependent activation of CooA is thus more complex than simple displacement of a ligand from the heme iron since NO does not trigger DNA binding. These observations suggest that the CooA heme site discriminates between NO and the biologically relevant signal, CO.

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Heme plays critical roles in numerous biological phenomena. Recent evidence has uncovered a new role of heme in cellular signal transduction, and its mechanism involves reversible binding of heme to proteins. This Account highlights the novel function of heme as an intracellular messenger in the regulation of gene expression and ion channel function.