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Heme binding to a conserved Cys-Pro-Val motif is crucial for the catalytic function of mitochondrial heme lyases
H Steiner1, G Kispal, A Zollner
1Institut für Physiologische Chemie, Physikalische Biochemie und Zellbiologie der Universität München, Goethestrasse 33, 80336 München, Federal Republic of Germany. lill@mailer.uni-marburg.de
Insights
Heme lyase enzymes directly bind heme, with the Cys-Pro-Val motif crucial for this interaction. This finding explains how heme is incorporated into essential mitochondrial proteins like cytochrome c1.
Area of Science:
- Biochemistry
- Molecular Biology
- Mitochondrial Function
Background:
- Mitochondrial cytochromes c and c1 require heme for function.
- Cytochrome c heme lyase (CCHL) and cytochrome c1 heme lyase (CC1HL) enzymes catalyze heme attachment.
- Both CCHL and CC1HL share sequence homology and a conserved Cys-Pro-Val (CPV) motif.
Purpose of the Study:
- To investigate direct heme binding by heme lyases.
- To determine the role of the CPV motif in heme binding and enzyme activity.
Main Methods:
- In vitro assays to assess heme binding to CC1HL.
- Analysis of protein conformation, refolding, and mitochondrial import.
- Site-directed mutagenesis of the CPV motif (CC1HLSPV and CC1HLDeltaCPV).
- Phenotypic analysis of yeast mutants expressing mutated CC1HL.
Main Results:
- Heme directly binds to CC1HL, stabilizing its conformation and promoting refolding.
- The CPV motif is essential for heme binding, as demonstrated by mutant studies.
- Mutant CC1HLSPV failed to bind heme, leading to accumulation of heme-free cytochrome c1 in yeast.
Conclusions:
- Heme lyases possess intrinsic heme-binding capabilities.
- The CPV motif is a critical component of the heme binding site in heme lyases.
- Understanding this interaction is key to elucidating heme incorporation into mitochondrial cytochromes.
Abstract:
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.