Heme binding to a conserved Cys-Pro-Val motif is crucial for the catalytic function of mitochondrial heme lyases.
Covalent attachment of heme to the apoforms of mitochondrial cytochromes c and c1 requires the activity of cytochrome c heme lyase (CCHL) and cytochrome c1 heme lyase (CC1HL), respectively. The two enzymes differ in their cytochrome specificity, but they are related in sequence, and both contain conserved Cys-Pro-Val (CPV) motifs. By using various in vitro assays we investigated whether heme can bind directly to heme lyases and whether the CPV motif may be involved in heme binding. Heme stabilized CC1HL, as a model protein, in a folded, protease-resistant conformation, stimulated the refolding of CC1HL after urea denaturation, and inhibited the import of the CC1HL precursor into mitochondria. These effects were not observed with a point mutant, CC1HLSPV, in which cysteine was replaced by serine, and with CC1HLDeltaCPV, in which the motif was deleted. These results show that heme lyases can bind heme directly, and they identify the CPV sequence as a structural element important for this interaction. The phenotype of a yeast mutant expressing CC1HLSPV is in good agreement with such a role of the CPV motif. The mutant cells accumulate the heme-free intermediate form of cytochrome c1 and display a severe deficiency in the holo form. We suggest that the CPV motif forms a crucial part of the substrate binding site for heme.
Steiner
H
H
Institut für Physiologische Chemie, Physikalische Biochemie und Zellbiologie der Universität München, Goethestrasse 33, 80336 München, Federal Republic of Germany. lill@mailer.uni-marburg.de
Kispal
G
G
Zollner
A
A
Haid
A
A
Neupert
W
W
Lill
R
R
D000595:Amino Acid Sequence; D001665:Binding Sites; D001692:Biological Transport; D003575:Cytochromes c1 / Q000378:metabolism; D005816:Genetic Complementation Test; D006418:Heme / Q000378:metabolism*; D008190:Lyases / Q000737:chemistry*; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D017510:Protein Folding; D011498:Protein Precursors / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D011498:Protein Precursors; D029701:Saccharomyces cerevisiae Proteins; D006418:Heme; D003575:Cytochromes c1; D008190:Lyases; C095893:CYT2 protein, S cerevisiae