Functional Divergence of Heme-Thiolate Proteins: A Classification Based on Spectroscopic Attributes


1. INTRODUCTION Heme proteins are among the most versatile players in the biological milieu; their functions range widely and include electron transfer, catalysis, and small-molecule sensing and transport.1 This broad functional diversity may be attributed to the protein environment that constitutes the heme cofactor’s binding site, particularly those amino acid residues that serve as axial ligands to the heme iron atom. For example, cytochromes, globins, heme oxygenases, and the majority of peroxidases employ the imidazole moiety of a histidine (His) residue as an axial ligand. Certain cytochromes are also coordinated by the thioether of methionine (Met) or, rarely, the polypeptide Nterminal amino group.2 Catalases utilize a tyrosine (Tyr) phenolate.3 An ever-growing class of heme proteins employs a cysteine (Cys) thiolate as an axial ligand to heme b (ironprotoporphyrin IX, Figure 1). Differences in solvent exposure, hydrophobicity, iron atom oxidation and spin state, and the identity of the iron atom axial ligand(s) give rise to a wide variety of spectroscopic characteristics. These spectroscopic signatures provide useful fingerprinting handles with which to classify Cys(thiolate)-ligated hemoproteins. Two distinct classes of heme thiolate proteins emerge, which cluster according to their biological functions. We call these two classes the type-1 and type-2 heme thiolates. Herein, we demonstrate how type-1 and type-2 heme thiolate proteins differ in coordination numbers, spin states, and propensities to undergo ligand switching when reduced. Type- 1 heme thiolates have a vacant or labile axial coordination site trans to the thiolate ligand, and they retain the thiolate ligand when reduced. The high-spin, five-coordinate Fe(II) heme is functionally essential in small-molecule activation. Type-2 heme thiolates are always low-spin with two axial ligands; however, the thiolate ligand is replaced upon heme reduction. A second ligand replacement at the low-spin, six-coordinate Fe(II) heme enables function in small-molecule sensing. The differences in spin and coordination states between type-1 and type-2 heme thiolates give rise to their distinctive spectroscopic properties, as described in sections 4−8, and correlate with their functions in small molecule activation or sensing, as described in sections 9−11. Because the spectroscopic signatures arise from variations in electronic structure at the heme cofactor, it follows that each type of Cys(thiolate)-ligated hemoprotein displays distinct reactivity. This difference in reactivity is then manifested in the two classes of thiolate-ligated hemoproteins: type-1 that retains its Cys- (thiolate) ligand throughout changes in the heme iron oxidation state and type-2 with a propensity to lose its Cys(thiolate) upon reduction of the heme iron (Figure 2). It is the differences in coordination behavior that give rise to the distinct spectroscopic signatures that allow their classification. Importantly, these two types of reactivity allow heme-thiolate proteins to play disparate roles in nature: as catalysts (type-1) or small-molecule sensors/ transporters (type-2). The purpose of this Review is to identify, separate, and classify these two types of heme-thiolate proteins on the basis of their distinct spectroscopic and functional properties. Because the classification system is based on spectroscopic attributes, and because the function is dependent on the distinctive reactivity arising from the heme electronic structure, spectroscopic and reactivity differences are presented before functional differences. This Review draws on nearly six decades of spectroscopic and functional data from well-studied and well-reviewed hemethiolate monooxygenases, but its new classification system is inspired by the emergence of new families of thiolate-ligated hemoproteins that function in numerous signaling pathways. Amazingly, these divergent and independent functions are possible despite the fact that each protein uses the exact same cofactor (heme b) and a common thiolate ligand in the firstcoordination sphere of the heme iron. Such diversity is made possible by utilizing the protein scaffold to modulate how the heme cofactor changes spin and coordination states upon substrate binding, gas binding, and/or changes in the redox state of the heme iron (Figure 2). This Review attempts to explain how nature utilizes the versatility of heme-thiolate proteins to play multiple indispensible roles in life processes.



Publication Date


Journal Title

Chemical Reviews


American Chemical Society

Digital Object Identifier (DOI)


Document Type