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Heme-CO as a probe of the conformational state of calmodulin
M C Marden1, L Leclerc, C Poyart
1INSERM U299, Hôpital de Bicêtre, France.
Insights
Calcium-activated calmodulin binds four heme-carbon monoxide (CO) molecules in hydrophobic pockets, not through iron-histidine bonds. This interaction is reversible and involves heterogeneous binding sites.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein-Ligand Interactions
Background:
- Calmodulin is a calcium-binding protein crucial for cellular signaling.
- Heme proteins, like hemoglobin, typically bind ligands via iron coordination.
Purpose of the Study:
- To investigate the interaction between heme-carbon monoxide (CO) and calmodulin in a calcium-dependent manner.
- To characterize the binding sites and kinetics of heme-CO on calmodulin.
Main Methods:
- Spectroscopic analysis, specifically monitoring the Soret absorption band shift.
- Photodissociation of CO from the complex.
- Kinetic studies of biomolecular recombination.
Main Results:
- Calmodulin binds four heme-CO molecules per protein only in the presence of calcium.
- Binding is indicated by a spectral shift from 407 nm to 420 nm, with evidence of heterogeneous binding sites.
- CO can be photodissociated, and recombination kinetics suggest a heterogeneous mixture.
- The complex does not reversibly bind oxygen.
- Heme association is likely through hydrophobic pockets, with a dissociation constant (Kd) of 0.5 microM.
Conclusions:
- Calmodulin's interaction with heme-CO is calcium-dependent and involves loosely associated hemes in hydrophobic pockets.
- Unlike hemoglobin, heme binding to calmodulin does not involve the iron atom coordinating with histidine.
- The binding sites are heterogeneous, opening upon calcium-induced protein conformational changes.
Abstract:
The interaction of heme-CO with calmodulin, in the presence of calcium, leads to a complex of four heme-CO molecules per protein. No interaction was observed in the absence of calcium. The binding of heme-CO to calmodulin was monitored by the shift in the Soret absorption band from 407 to 420 nm (bound form); the four sites are not spectrally identical. The ligand CO can be photodissociated from the calmodulin-heme-CO complex and the biomolecular recombination kinetics also indicate a heterogeneous mixture. The complex does not bind oxygen reversibly. As calmodulin has only one histidine, the hemes are apparently not bound by the iron atom as in hemoglobin, but are probably loosely associated (Kd = 0.5 microM) in hydrophobic pockets which apparently open when the protein is activated by calcium.