Heme-CO binding to tryptophan-containing calmodulin mutants.
The binding of heme-CO to genetically engineered calmodulin containing a single tryptophan residue has been studied. A tryptophan residue was integrated at one of five positions: 26 or 62 of the N-terminal, 81 in the central helix, or 99 or 135 of the C-terminal. As for the wild type, the mutant calmodulins bind four molecules of heme-CO with an average affinity of 1 microM. (i) Homotropic effect. The quenching of the tryptophan fluorescence by energy transfer to the hemes indicates that there is no preference between the N- or C-terminal pockets for heme binding. The quenching is less than expected for a binomial distribution of four sites. This could indicate a lower energy transfer rate due to a specific orientation factor. The weak quenching as a function of the number of hemes bound may also reveal a cooperativity in the heme binding; the data can be simulated assuming two pairs of sites, where each pocket shows a cooperative binding for two hemes. (ii) Heterotropic effect. As observed for the wild type, addition of melittin does not displace the hemes from the mutant calmodulins; the affinity of heme-CO for the calmodulin.melittin complex is higher than that for calmodulin alone. The affinity of heme-CO for native calmodulin is also higher in the presence of trifluoperazine.
Institut National de la Santé et de la Recherche Médicale, Unité 299, Hôpital de Bicêtre, Le Kremlin-Bicêtre, France.
E
E
Leclerc L'Hostis
L
L
Leclerc
J
J
Haiech
C
C
Poyart
M C
MC
Marden
D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism*; D002248:Carbon Monoxide / Q000737:chemistry*; D002417:Cattle; D004735:Energy Transfer; D006418:Heme / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D014268:Trifluoperazine / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry*
D002147:Calmodulin; D014268:Trifluoperazine; D006418:Heme; D002248:Carbon Monoxide; D014364:Tryptophan