Regulation of the Heme A Biosynthetic Pathway

Abstract

The assembly and activity of cytochrome c oxidase is dependent on the availability of heme A, one of its essential cofactors. In eukaryotes, two inner mitochondrial membrane proteins, heme O synthase (Cox10) and heme A synthase (Cox15), are required for heme A biosynthesis. In this report, we demonstrate that in Saccharomyces cerevisiae the transcription of COX15 is regulated by Hap1, a transcription factor whose activity is positively controlled by intracellular heme concentration. Conversely, COX10, the physiological partner of COX15, does not share the same regulatory mechanism with COX15. Interestingly, protein quantification identified an 8:1 protein ratio between Cox15 and Cox10. Together, these results suggest that heme A synthase and/or heme O synthase might play a new, unidentified role in addition to heme A biosynthesis.The biosynthesis of heme A, an obligatory cofactor of cytochrome c oxidase (CcO), 2 requires two nuclear-encoded proteins, heme O synthase (HOS) and heme A synthase (HAS) (1-5). Like CcO, both HOS and HAS are integral membrane proteins localized to the inner mitochondrial membrane. First, heme O synthase catalyzes the conversion of heme B to heme O via the farnesylation of a vinyl group at position C-2 (5). Second, heme A synthase converts heme O to heme A by oxidizing the C-8 methyl substituent on heme O to an aldehyde group (1, 2). Heme A has no known physiological function other than as a cofactor in the terminal heme-copper oxidases.COX10 and COX15, encoding eukaryotic HOS and HAS, respectively, are functionally conserved from yeast to humans (6, 7). In yeast, heme A deficiency leads to respiratory incompetence and the degradation of CcO (8). Mutations in either COX10 or COX15 in humans lead to metabolic diseases associated with isolated CcO deficiency, including Leigh syndrome and hypertrophic cardiomyopathy (9, 10). Recently, a deficiency in heme A biosynthesis was suggested to be involved in age-related decline of CcO in patients with Alzheimer disease (11).Assembly of the multisubunit CcO complex is a complicated and sequential process (12) that requires more than two dozen nuclear-encoded accessory proteins (13). The crystal structure of bovine heart CcO reveals that the two heme A molecules are deeply buried in the hydrophobic pocket of CcO (14), implying that the insertion of heme A probably occurs early during the CcO assembly process. The identification of a heme a-Cox1 intermediate and the lack of Cox1-Cox4-Cox5 subassembly complex in COX10-deficient human cells further support the notion that heme A insertion occurs at an early step during CcO assembly (12,15,16). In Rhodobacter sphaeroides, a deletion of Surf1 results in a 50% loss of heme a 3 , indicating that Surf1 might be involved in heme a 3 maturation (17).Because the availability of heme A is a key determinant in the assembly of CcO, the reactions catalyzed by HOS and HAS are likely targets for regulation. Because of its high reduction potential (18), free heme A is potentially even more toxic than free heme B. Th...

Surf1, Associated with Leigh Syndrome in Humans, Is a Heme-binding Protein in Bacterial Oxidase Biogenesis

“…Therefore, in eukaryotes, a direct heme a transfer from COX15 to COX SU I was assumed. COX15 activity and expression were found to be highly regulated in yeast, especially by heme b, which is not only a precursor of heme a but also the cofactor of heme a synthase (38). In the same study, it was proposed that heme a liberation from COX15 could be regulated by SU I or by COX assembly intermediates because this would prevent uncontrolled release of potentially toxic heme a.…”

From Synthesis to Utilization: The Ins and Outs of Mitochondrial Heme

“…Identification of the N196K substitution led to the conclusion that Cox10 activation may be linked to a multimeric structure. Because Cox10 is present at approximately 8-fold lower abundance than the heme a synthase enzyme discussed below, the synthesis of heme o is likely the rate-limiting step of heme a production [135].…”

“…In contrast to Cox10, the Cox15 active site is predicted to reside on the IMS side of the IMM; no information is available as to how heme o is transported across the IM to the Cox15 active site. Another riddle to this is the fact that eukaryotic Cox10 and Cox15 do not stably interact [132] and are differentially regulated [135,139]. Unlike COX10, COX15 gene expression is regulated by the transcription factor Hap1, which is itself regulated by heme concentration [140].…”

“…Unlike COX10, COX15 gene expression is regulated by the transcription factor Hap1, which is itself regulated by heme concentration [140]. As such, Cox15 protein levels are strictly regulated by heme availability [135,139]. It has been suggested by studies of the bacterial Cox15 that the protein contains a heme b cofactor [106], however, whether this proposed cofactor is necessary for enzymatic function or maintaining protein structure has yet to be determined.…”

Structure, function, and assembly of heme centers in mitochondrial respiratory complexes

“…This connection of heme a biosynthesis and CcO biogenesis may serve regulatory purposes to minimize excess and toxic levels of highly reactive heme a . Cox10-catalyzed conversion of protoheme appears to be a rate-limiting step in heme a formation, given its ~8-fold dearth over Cox15 in yeast mitochondria [94]. It is therefore likely that regulation of Cox10 abundance/activity is the way to modulate the heme a biosynthetic pathway.…”