Heme Oxygenase: Species Variation
Members of the heme oxygenase family, in particular HO-1 and HO-2, are key enzymes in heme catabolism. HO-1 and HO-2 are encoded by single-copy genes in the human (HMOX1, HMOX2) and mouse genome (Hmox1, Hmox2) 150. The HMOX1 gene is conserved in mammals including chimpanzee, Rhesus monkey, dog, cow, mouse, and rat; in chicken and zebrafish (Danio rerio) as well as in frog. 228 organisms have orthologs with the human HMOX1 gene. The coding sequence of the expressed HO-1 protein consists of 288 amino acid residues yielding a molecular mass of 32.8 kDa. The HMOX2 gene is conserved in all of the organisms mentioned above, and orthologs of the human HOMX2 gene can be found in 233 organisms. Unlike HO-1, human HO-2 bears additional 30 amino acid residues at the N-terminus as well as two Cys-Pro dipeptides near the C-terminus yielding a protein with 316 amino acids and a molecular mass of 36 kDa (Figure 3). A third isoform, termed HO-3 has been described in rats only 40. As the expression of HO-3 could not be determined at both, the mRNA and the protein level in rats, HO-3 is considered as being a pseudogene (HO3; Hmox2-ps1) that originates from the HMOX2 mRNA 40. Recently, a novel splice variant of human HO-1, harboring a 164-aa deletion (aa49 – 212 of 32 kDa HO-1) and a molecular mass of 14 kDa, was identified in malignant cells 6. HO-1 is ubiquitously present in higher animals showing a high structural similarity and functional identity in humans, large mammals, rodents, and birds. Of note, numerous HO-1-like proteins and activities have been reported in lower organisms such as algae, bacteria, fungi, flies, and plants.
Although several organisms have been found to express HOs, there is divergence with respect to structural and functional properties reﬂecting a diversity of responsibility. Based on these characteristics, two groups of HOs have been classified. The first discovered group of HOs is the HO-1 subfamily comprising canonical HOs that are present in both, prokaryotes and eukaryotes including, e.g., mammalian HO-1/-2 as well as microbial HmuO and PigA/HemO 1, 2, 41, 57, 67, 84. HO-like molecules from bacteria utilize heme as a preferred nutritional iron source under low-iron conditions 151. As a result, expression of bacterial HO-like enzymes is up- or down-regulated in relation to the iron concentration 151. Members of the HO-1 subfamily belong to the alpha-only protein class and share a common secondary structure, bearing nine to ten α-helices and a highly conserved catalytic site in the cytoplasmic domain 76. They degrade heme to biliverdin IXα, carbon monoxide (CO), and ferrous iron (Fe2+) 1. The second group of HOs which can predominantly be found in a limited number of bacterial species contains non-canonical and IsdG-like HOs (e.g., IsdG/l from S. aureus and MuhD from M. tuberculosis), converting heme to metabolites different from biliverdin and CO, such as staphylobilin, formaldehyde and mycobilin 48, 87, 88. Several members of the IsdG subfamily are selectively expressed under low-iron conditions. The founding members of this group, IsdG and its paralog IsdI from S. aureus, show 64% sequence identity and 79% sequence similarity converting heme to staphylobilin and formaldehyde 48, 88. IsdG/I and also MuhD from M. tuberculosis belong to the class of α + β proteins as part of the dimeric α + β-barrel superfamily that dimerize across their β-sheets 89. MuhD shows 24% sequence identity and 46% sequence similarity to IsdG. IsdG/I and MhuD bear a ferredoxin-like fold in their ABM domain, formed by three α-helices and four β-strands organized in a βαββααβ pattern. His77 in IsdG and His75 in MuhD were identified as crucial amino acid residues involved in heme ligation 87, 152. In E. coli, the IsdG subfamily member ChuW has been reported to anaerobically degrade heme to the small molecule anaerobilin and free iron 90. It is noteworthy that E. coli expresses a second HO-like enzyme, ChuS, which catalyzes the conversion of heme to biliverdin and CO 83. The structure of ChuS is unique as it contains a structural duplication, consisting of ten α-helices and 18 β-strands arranged in a way that enables the characteristic formation of the enzyme core from two β-sheets of nine anti-parallel β-strands 83. The enzyme core is surrounded by three α-helices organized in a C-terminal α-loop-α-loop-α motif and an N-terminal pair of parallel α-helices. The C- and N-termini show only limited primary sequence homology but exhibit structural duplication in its tertiary structure 83. Bacterial HO-like enzymes have also been identified in Corynebacterium spec. (HmuO) 41, 42, Neisseria meningitidis and N. gonorrhoeae (HemO) 43, 44, Yersinia enterocolitica (HemS) 153, and Pseudomonas aeruginosa (PhuS) 154. HemS shares 35% sequence identity and 51% similarity to PhuS, and 34% sequence identity and 49% similarity to ChuS 89. Comparibly to S. aureus, P. aeruginosa encodes multiple HO variants such as PigA/HemO, PhuS, and BphO. BphO of P. aeruginosa produces exclusively α-biliverdin for the synthesis of bacterial phytochrome. PigA/HemO is somewhat different from other HO-1 subfamily members as it generates a 3 : 7 mixture of β- and δ-biliverdin 155. The β-/δ-biliverdin ratio has been noted to depend on the nature of aa189 (Phe189) as a Phe189Trp mutation in PigA/HemO predominantly generates β-biliverdin, indicative for an aa189-heme interaction affecting enzyme topology and reaction regioselectivity 89. In contrast to HO-1 and HumO, PigA/HemO harbors a characteristic heme rotation within the heme-binding pocket which assigns the δ-meso heme carbon to the same position as the α-meso heme carbon in other HOs 155. While only PigA/HemO is directly involved in heme iron utilization 84, BphO catalyzes the formation of biliverdin required for the assembly of the phytochrome-like photoreceptor BphP 85. It is worth mentioning that BphO is expressed independently of the iron status but its expression is regulated by the cell density 89. Several of the above mentioned microbial HOs show a high degree of homology to mammalian HO-1 141. Structurally, HemS from Y. enterocolitica and ChuS from E. coli act as monomers 78, 79, while PhuS from P. aeruginosa crystalizes as a dimer 80, 81. However, the monomeric form has been identified as the dominant form in solution showing a significantly higher stability than the dimeric variant 80, 81. Crystallized PhuS dimers have been proposed to align across their β-sheets, thereby displaying a structure highly comparable to ChuS 89. PhuS consists of three α-helices organized in a C-terminal α-loop-α-loop-α motif and an N-terminal pair of parallel α-helices 89. In contrast to ChuS, the binding pocket in PhuS and HemS is more accessible, with the C-terminal α-helices being more adjacent to the binding site 156, 157. Together with recent data, these findings imply that PhuS acts as an intracellular heme transport protein in vivo 158 which guides heme to PigA/HemO in P. aeruginosa 159.
Up to date, microbial HO-like enzymes have been recognized in more than 4,000 bacterial species, >500 fungal species, and a low number of archaeal species. With respect to fungi, HOs are expressed in pathogenic as well as non-pathogenic organisms. The HO ortholog Hmx1p was identified in S. cerevisiae as a stress protein in response to iron deprivation 45. Iron uptake is predominantly regulated by the constitutively expressed cytosolic transcription factor Atf1p which translocates to the nucleus upon iron deprivation where it triggers transcription of target genes such as HMX1 160. Apart from iron starvation, several other stressors were recently identified by Collinson and colleagues able to induce the expression of Hmx1p 161. The authors provide evidence for a further role of Hmx1p in cellular response and anti-oxidant defense. The anti-oxidant properties could be attributed to the up-regulation of several well-established anti-oxidant defense enzyme genes, including GSH1, GPX1, CTT1, and MXR1. Notably, the studies by Collinson et al. also identified numerous HO-dependent, differentially expressed genes whose products play crucial roles in several previously unrecognized mechanisms, including RNA processing, ribosome biogenesis, transcriptional regulation, and membrane transport 161. As only a minor percentage of differentially expressed genes contribute to anti-oxidant defense in yeast, anti-oxidant protection might represent a secondary function of Hmx1p in this organism. Thus, Hmx1p more likely regulates cellular and biological functions irrespective of its enzymatic activity. Unlike S. cerevisiae, the pathogenic fungus Candida albicans uses extracellular hemin as iron source 46. The CaHMX1 gene encodes a functional HO enzyme exclusively catalyzing the synthesis of the α-isomer of biliverdin 162. C. albicans features an hemoglobin (Hb) receptor which links Hb exposure to intracellular signaling cascades, thereby triggering the expression of various genes 163, 164. Hb-induced CaHMX1 transcription also takes place under iron abundance which may be present in human tissues during advanced infections. Since Hb represents a plenteous iron source in a mammalian host, this response might be considered as being an adaptation of the CaHMX1 gene regulation aiming at promoting iron acquisition from the host. It is therefore likely that CaHMX1 may fulfill other duties apart from the usage of heme and Hb as nutritional iron sources 46, 165.
Plants express several HOs but these often lack the C-terminal transmembrane segment (TMS) found in mammalian HOs. Plant HOs, that are more closely related to bacterial and insect HO-1 than to mammalian HO-2, catalyze the oxidative degradation of heme to biliverdin IXα, CO, and Fe2+. HOs in plants are also crucially involved in cellular defense, stomatal regulation, iron mobilization, and lateral root formation. Additionally, plant HOs play an essential role in photomorphogenesis as biliverdin IXα serves as an important intermediate in the phytochrome chromophore biogenesis (for a review see Mahawar and Shekhawat, 2018) 166. The model plant A. thaliana possesses three biochemically indistinguishable functional HO proteins (AtHO-1, AtHO-3, AtHO-4) and one probably inactive HO (AtHO-2) 10. AtHO-1, AtHO-3, and AtHO-4 are active monomeric HOs able to convert heme to biliverdin IXα 10. All four proteins are encoded in the nucleus but contain chloroplast translocation sequences at their N-termini sufficient for chloroplast translocation. Two isoforms as the result of alternative splicing have been described for AtHO-1. The shorter isoform differs from the canonical sequence by an aa94 – 142 → D substitution. Notwithstanding the above, no experimental confirmation is available for the shorter isoform apart from the canonical sequence. Four isoforms have been reported for AtHO-2: one described alternatively spliced isoform 299 amino acids in length and three potential isoforms that are computationally mapped. Two isoforms produced by alternative splicing have been noted for AtHO-3. Isoform 2 of AtHO-3 bears a VSKKILDN → LCRYLRRY substitution at position 220 – 227 and lacks the amino acid stretch 228 – 285 of the canonical sequence. Of note, no experimental confirmation is available for the shorter isoform apart from the canonical sequence.
From the unicellular green alga C. reinhardtii, two HO members (HO-1 and HO-2) have been isolated 167. The Chlamydomonas HO-1 gene (HMOX1) has been well characterized, and its expression is induced by numerous stimuli. The second gene (HMOX2) which is differently expressed shows divergent sequence similarity to HMOX1. A third putative enzyme termed Lfo-1 has recently been identified, bearing an antibiotic biosynthesis mono-oxygenase (ABM) domain commonly found in IsdG subfamily members 11. This HO-like protein aerobically degrades heme to a distinct unidentified heme metabolite, its predicted secondary structure resembles those of the IsdG subfamily members, and it harbors the functionally conserved catalytic residues found in all HOs of the IsdG subfamily 11. Lfo-1 is encoded by a nuclear gene (Cre07.g31230) that is more highly expressed under iron starvation conditions than under iron saturation conditions 168. The coding sequence of the expressed protein consists of 171 amino acid residues yielding a molecular mass of approximately 18 kDa 11, 167. Functional characterization of Lfo-1 revealed that this type of heme degradation depends on the presence of three catalytic amino acid residues that are conserved amongst the IsdG subfamily members 11.
HO molecules act as central players in the biosynthesis of the chromophoric part of the photosynthetic antennae in cryptophyceae, cyanobacteria, and red algae. Unlike mammalian HOs that are associated with the microsomal cell fraction, HO from the red alga Cyanidium caldarium is a 38 kDa soluble enzyme that can be induced by several heme precursors and which requires ferredoxin and ferredoxin-NADP reductase for activity 169, 170. Amongst others, HO-encoding genes (pbsA) were isolated and characterized from the chloroplast genome of Porphyra purpurea 171 and Rhodella violacea 13. Of note, the HO gene from C. caldarium is located to the nucleus while protein translation occurs at cytosolic ribosomes 169. Two distinct HO genes, termed ho, are present in the cyanobacterium Synechocystis sp. strain PCC6803 as evidenced by homology analyses using P. purpurea pbsA for comparison 172. It is noteworthy that pbsA from R. violacea is devided into three exons whilst pbsA from P. purpurea is continuous, highlighting the existence of split genes in the rhodophyte plastid genome 13. As the expression of pbsA from R. violacea is transcriptionally activated under iron limitation, a putative additional function for HO as an iron-mobilizing agent in photosynthetic organisms should be taken into account 13. The amino acid sequence of R. violacea PbsA shares identity of 76%, 73%, and 65% with P. purpurea, Synechocystis sp. strain PCC6803 (product of gene 1, sll1184), and Synechocystis sp. strain PCC6803 (product of gene 2, sll1875), respectively. The predicted molecular mass of R. violacea PbsA is 27 kDa and thus markedly lower than that of the 38 kDa heme-binding molecule of C. caldarium 170. Notwithstanding the above, the active centers of rhodophyte HOs are highly conserved and bear an essential histidyl residue at position 154 required for heme ligation 173.
In view of the above, the divergent subcellular location of HO-encoding genes in rhodophytes can give insight into evolutionary processes. As already mentioned, the HO gene from C. caldarium is located to the nucleus while the corresponding genes in P. purpurea and R. violacea are of chloroplastic origin. From these findings one can speculate that the export of genes from the chloroplast to the nucleus is less abundant in P. purpurea and R. violacea than in Cyanidiophyceae. Together with the nuclear location of the corresponding genes in the green alga, C. reinhardtii, and in higher plants, a manifold organelle-specific role of HO becomes apparent. While in pathogenic bacteria and fungi HO obviously plays a pivotal role in iron utilization from exogenous heme or hemoglobin during host infection 42, 151, animals use HOs for several physiological processes beside heme degradation including, e.g., anti-oxidant response as well as modulation of cell growth and differentiation 174. In photosynthetically active organisms (plants, algae, cyanobacteria), HO could play an ambivalent role, on the one hand by providing the first step in the biosynthesis of chromophores and photoreceptors (phytochromobillins, phycobillins, bacterial phytochromes) and, on the other hand, in the release of iron from convenient cell compounds under low-iron conditions 13, 151.
Only limited information is available on the structure and function of archaeal HOs. Up to date, HOs could be identified in a small number of archaeal species, including IsdG from the human-associated halophilic archaeon Haloferax massiliensis, and Ho from Euryarchaeota archaeon.