Cloning and analysis of the Neurospora crassa gene for cytochrome c heme lyase

The cyt-2-1 mutant of Neurospora crassa is deficient in cytochromes aa3 and c and in cytochrome c heme lyase activity (Mitchell, M.B., Mitchell, H.K., and Tissieres, A. (1953) Proc. Natl. Acad. Sci. U.S.A. 39, 606-613; Nargang, F.E., Drygas, M.E., Kwong, P.L., Nicholson, D.W., and Neupert, W. (1988) J. Biol. Chem. 263, 9388-9394). By rescue of the slow growth character of the cyt-2-1 mutant, we have cloned the cyt-2+ gene from a N. crassa genomic library using sib selection. Analysis of the DNA sequence of the cyt-2+ gene revealed an open reading frame of 346 amino acids that has homology to the yeast cytochrome c heme lyase. The open reading frame is interrupted by two short introns. Codon usage and Northern hybridization analysis suggest that the cyt-2 gene is expressed at low levels. The cyt-2-1 mutant allele was cloned from a partial cyt-2-1 gene bank using the wild-type gene as a probe. Sequence analysis of the mutant gene revealed a 2-base (CT) deletion that alters the reading frame for 21 codons before generating an early stop codon in the protein-coding sequence. It was previously suggested that the cyt-2-1 mutation inactivates one of two regulatory circuits controlling the production of cytochrome aa3. The finding that the cyt-2-1 mutation affects the coding sequence for cytochrome c heme lyase provides a direct explanation for the deficiency of cytochrome c in the mutant and suggests that the lack of cytochrome aa3 is a regulatory response to the deficiency of cytochrome c.     

Role of cytochrome c heme lyase in the import of cytochrome c into mitochondria

The import of cytochrome c into Neurospora crassa mitochondria was examined at distinct stages in vitro. The precursor protein, apocytochrome c, binds to mitochondria with high affinity and specificity but is not transported completely across the outer membrane in the absence of conversion to holocytochrome c. The bound apocytochrome c is accessible to externally added proteases but at the same time penetrates far enough through the outer membrane to interact with cytochrome c heme lyase. Formation of a complex in which apocytochrome c and cytochrome c heme lyase participate represents the rate-limiting step of cytochrome c import. Conversion from the bound state to holocytochrome c, on the other hand, occurs 10-30-fold faster. Association of apocytochrome c with cytochrome c heme lyase also takes place after solubilizing mitochondria with detergent. We conclude that the bound apocytochrome c, spanning the outer membrane, forms a complex with cytochrome c heme lyase from which it can react further to be converted to holocytochrome c and be translocated completely into the intermembrane space.     

Role of cytochrome c heme lyase in mitochondrial import and accumulation of cytochrome c in Saccharomyces cerevisiae.

Heme is covalently attached to cytochrome c by the enzyme cytochrome c heme lyase. To test whether heme attachment is required for import of cytochrome c into mitochondria in vivo, antibodies to cytochrome c have been used to assay the distributions of apo- and holocytochromes c in the cytoplasm and mitochondria from various strains of the yeast Saccharomyces cerevisiae. Strains lacking heme lyase accumulate apocytochrome c in the cytoplasm. Similar cytoplasmic accumulation is observed for an altered apocytochrome c in which serine residues were substituted for the two cysteine residues that normally serve as sites of heme attachment, even in the presence of normal levels of heme lyase. However, detectable amounts of this altered apocytochrome c are also found inside mitochondria. The level of internalized altered apocytochrome c is decreased in a strain that completely lacks heme lyase and is greatly increased in a strain that overexpresses heme lyase. Antibodies recognizing heme lyase were used to demonstrate that the enzyme is found on the outer surface of the inner mitochondrial membrane and is not enriched at sites of contact between the inner and outer mitochondrial membranes. These results suggest that apocytochrome c is transported across the outer mitochondrial membrane by a freely reversible process, binds to heme lyase in the intermembrane space, and is then trapped inside mitochondria by an irreversible conversion to holocytochrome c accompanied by folding to the native conformation. Altered apocytochrome c lacking the ability to have heme covalently attached accumulates in mitochondria only to the extent that it remains bound to heme lyase.     

Biogenesis of cytochrome c1. Role of cytochrome c1 heme lyase and of the two proteolytic processing steps during import into mitochondria

The biogenesis of cytochrome c1 involves a number of steps including: synthesis as a precursor with a bipartite signal sequence, transfer across the outer and inner mitochondrial membranes, removal of the first part of the presequence in the matrix, reexport to the outer surface of the inner membrane, covalent addition of heme, and removal of the remainder of the presequence. In this report we have focused on the steps of heme addition, catalyzed by cytochrome c1 heme lyase, and of proteolytic processing during cytochrome c1 import into mitochondria. Following translocation from the matrix side to the intermembrane-space side of the inner membrane, apocytochrome c1 forms a complex with cytochrome c1 heme lyase, and then holocytochrome c1 formation occurs. Holocytochrome c1 formation can also be observed in detergent-solubilized preparations of mitochondria, but only after apocytochrome c1 has first interacted with cytochrome c1 heme lyase to produce this complex. Heme linkage takes place on the intermembrane-space side of the inner mitochondrial membrane and is dependent on NADH plus a cytosolic cofactor that can be replaced by flavin nucleotides. NADH and FMN appear to be necessary for reduction of heme prior to its linkage to apocytochrome c1. The second proteolytic processing of cytochrome c1 does not take place unless the covalent linkage of heme to apocytochrome c1 precedes it. On the other hand, the cytochrome c1 heme lyase reaction itself does not require that processing of the cytochrome c1 precursor to intermediate size cytochrome c1 takes place first. In conclusion, cytochrome c1 heme lyase catalyzes an essential step in the import pathway of cytochrome c1, but it is not involved in the transmembrane movement of the precursor polypeptide. This is in contrast to the case for cytochrome c in which heme addition is coupled to its transport directly across the outer membrane into the intermembrane space.     

Import of cytochrome c into mitochondria. Cytochrome c heme lyase

The import of cytochrome c into mitochondria can be resolved into a number of discrete steps. Here we report on the covalent attachment of heme to apocytochrome c by the enzyme cytochrome c heme lyase in mitochondria from Neurospora crassa. A new method was developed to measure directly the linkage of heme to apocytochrome c. This method is independent of conformational changes in the protein accompanying heme attachment. Tryptic peptides of [35S]cysteine-labelled apocytochrome c, and of enzymatically formed holocytochrome c, were resolved by reverse-phase HPLC. The cysteine-containing peptide to which heme was attached eluted later than the corresponding peptide from apocytochrome c and could be quantified by counting 35S radioactivity as a measure of holocytochrome c formation. Using this procedure, the covalent attachment of heme to apocytochrome c, which is dependent on the enzyme cytochrome c heme lyase, could be measured. Activity required heme (as hemin) and could be reversibly inhibited by the analogue deuterohemin. Holocytochrome c formation was stimulated 5--10-fold by NADH greater than NADPH greater than glutathione and was independent of a potential across the inner mitochondrial membrane. NADH was not required for the binding of apocytochrome c to mitochondria and was not involved in the reduction of the cysteine thiols prior to heme attachment. Holocytochrome c formation was also dependent on a cytosolic factor that was necessary for the heme attaching step of cytochrome c import. The factor was a heat-stable, protease-insensitive, low-molecular-mass component of unknown function. Cytochrome c heme lyase appeared to be a soluble protein located in the mitochondrial intermembrane space and was distinct from the previously identified apocytochrome c binding protein having a similar location. A model is presented in which the covalent attachment of heme by cytochrome c heme lyase also plays an essential role in the import pathway of cytochrome c.     

Coupling of heme attachment to import of cytochrome c into yeast mitochondria. Studies with heme lyase-deficient mitochondria and altered apocytochromes c

Cytochrome c is synthesized in the cytoplasm as apocytochrome c, lacking heme, and then imported into mitochondria. The relationship between attachment of heme to the apoprotein and its import into mitochondria was examined using an in vitro system. Apocytochrome c transcribed and translated in vitro could be imported with high efficiency into mitochondria isolated from normal yeast strains. However, no import of apocytochrome c occurred with mitochondria isolated from cyc3- strains, which lack cytochrome c heme lyase, the enzyme catalyzing covalent attachment of heme to apocytochrome c. In addition, amino acid substitutions in apocytochrome c at either of the 2 cysteine residues that are the sites of the thioether linkages to heme, or at an immediately adjacent histidine that serves as a ligand of the heme iron, resulted in a substantial reduction in the ability of the precursor to be translocated into mitochondria. Replacement of the methionine serving as the other iron ligand, on the other hand, had no detectable effect on import of apocytochrome c in this system. Thus, covalent heme attachment is a required step for import of cytochrome c into mitochondria. Heme attachment, however, can occur in the absence of mitochondrial import since we have detected CYC3-encoded heme lyase activity in solubilized yeast extracts and in an Escherichia coli expression system. These results suggest that protein folding triggered by heme attachment to apocytochrome c is required for import into mitochondria.     

Overlapping specificities of the mitochondrial cytochrome c and c1 heme lyases

Heme attachment to the apoforms of fungal mitochondrial cytochrome c and c1 requires the activity of cytochrome c and c1 heme lyases (CCHL and CC1HL), which are enzymes with distinct substrate specificity. However, the presence of a single heme lyase in higher eukaryotes is suggestive of broader substrate specificity. Here, we demonstrate that yeast CCHL is active toward the non-cognate substrate apocytochrome c1, i.e. CCHL promotes low levels of apocytochrome c1 conversion to its holoform in the absence of CC1HL. Moreover, that the single human heme lyase also displays a broader cytochrome specificity is evident from its ability to substitute for both yeast CCHL and CC1HL. Multicopy and genetic suppressors of the absence of CC1HL were isolated and their analysis revealed that the activity of CCHL toward cytochrome c1 can be enhanced by: 1) reducing the abundance of the cognate substrate apocytochrome c, 2) increasing the accumulation of CCHL, 3) modifying the substrate-enzyme interaction through point mutations in CCHL or cytochrome c1, or 4) overexpressing Cyc2p, a protein known previously only as a mitochondrial biogenesis factor. Based on the functional interaction of Cyc2p with CCHL and the presence of a putative FAD-binding site in the protein, we hypothesize that Cyc2p controls the redox chemistry of the heme lyase reaction.     

Import of apocytochrome c into the mitochondrial intermembrane space along a cytochrome c1 sorting pathway

The question of whether cytochrome c could be functionally sorted to the mitochondrial intermembrane space along a "conservative sorting" pathway was investigated using a fusion protein termed pLc1-c. pLc1-c contains 3-fold targeting information, namely, the complete bipartite presequence of the cytochrome c1 precursor joined to the amino terminus of apocytochrome c. pLc1-c could be selectively imported into the intermembrane space either directly across the outer membrane along a cytochrome c import route or along a cytochrome c1 route via the matrix. Thus, apocytochrome c could be sorted along a conservative sorting pathway; however, following reexport from the matrix, apo-Lc1-c could not be converted to its holo counterpart. Despite the apparent similarity of structure and functional location of the heme lyases and similarity of the heme binding regions in their respective apoproteins, cytochrome c heme lyase and cytochrome c1 heme lyase apparently have different and nonoverlapping substrate specificities.     

Accumulation of pre-apocytochrome f in a Synechocystis sp. PCC 6803 mutant impaired in cytochrome c maturation.

Cytochrome c maturation involves heme transport and covalent attachment of heme to the apoprotein. The 5' end of the ccsB gene, which is involved in the maturation process and resembles the ccs1 gene from Chlamydomonas reinhardtii, was replaced by a chloramphenicol resistance cartridge in the cyanobacterium Synechocystis sp. PCC 6803. The resulting Delta(M1-A24) mutant lacking the first 24 ccsB codons grew only under anaerobic conditions. The mutant retained about 20% of the wild-type amount of processed cytochrome f with heme attached, apparently assembled in a functional cytochrome b(6)f complex. Moreover, the mutant accumulated unprocessed apocytochrome f in its membrane fraction. A pseudorevertant was isolated that regained the ability to grow under aerobic conditions. The locus of the second-site mutation was mapped to ccsB, and the mutation resulted in the formation of a new potential start codon in the intergenic region, between the chloramphenicol resistance marker and ccsB, in frame with the remaining part of ccsB. In this pseudorevertant the amount of holocyt f increased, whereas that of unprocessed apocytochrome f decreased. We suggest that the original deletion mutant Delta(M1-A24) expresses an N-terminally truncated version of the protein. The stable accumulation of unprocessed apocytochrome f in membranes of the Delta(M1-A24) mutant may be explained by its association with truncated and only partially functional CcsB protein resulting in protection from degradation. Our attempt to delete the first 244 codons of ccsB in Synechocystis sp. PCC 6803 was not successful, suggesting that this would lead to a lack of functional cytochrome b(6)f complex. The results suggest that the CcsB protein is an apocytochrome chaperone, which together with CcsA may constitute part of cytochrome c lyase.     

The mitochondrial cytochrome c N-terminal region is critical for maturation by holocytochrome c synthase

The covalent attachment of heme to mitochondrial cytochrome c is catalysed by holocytochrome c synthase (HCCS, also called heme lyase). How HCCS functions and recognises the substrate apocytochrome is unknown. Here we have examined HCCS recognition of a chimeric substrate comprising a short mitochondrial cytochrome c N-terminal region with the C-terminal sequence, including the CXXCH heme-binding motif, of a bacterial cytochrome c that is not otherwise processed by HCCS. Heme attachment to the chimera demonstrates the importance of the N-terminal region of the cytochrome. A series of variants of a mitochondrial cytochrome c with amino acid replacements in the N-terminal region have narrowed down the specificity determinants, providing insight into HCCS substrate recognition.