Role of heme iron coordination and protein structure in the dynamics and geminate rebinding of nitric oxide to the H93G myoglobin mutant: implications for nitric oxide sensors.
The influence of the heme iron coordination on nitric oxide binding dynamics was investigated for the myoglobin mutant H93G (H93G-Mb) by picosecond absorption and resonance Raman time-resolved spectroscopies. In the H93G-Mb, the glycine replacing the proximal histidine does not interact with the heme iron so that exogenous substituents like imidazole may coordinate to the iron at the proximal position. Nitrosylation of H93G-Mb leads to either 6- or 5-coordinate species depending on the imidazole concentration. At high concentrations, (imidazole)-(NO)-6-coordinate heme is formed, and the photoinduced rebinding kinetics reveal two exponential picosecond phases ( approximately 10 and approximately 100 ps) similar to those of wild type myoglobin. At low concentrations, imidazole is displaced by the trans effect leading to a (NO)-5-coordinate heme, becoming 4-coordinate immediately after photolysis as revealed from the transient Raman spectrum. In this case, NO rebinding kinetics remain bi-exponential with no change in time constant of the fast component whose amplitude increases with respect to the 6-coordinate species. Bi-exponential NO geminate rebinding in 5-coordinate H93G-Mb is in contrast with the single-exponential process reported for nitrosylated soluble guanylate cyclase (Negrerie, M., Bouzhir, L., Martin, J. L., and Liebl, U. (2001) J. Biol. Chem. 276, 46815-46821). Thus, our data show that the iron coordination state or the heme iron out-of-plane motion are not at the origin of the bi-exponential kinetics, which depends upon the protein structure, and that the 4-coordinate state favors the fast phase of NO geminate rebinding. Consequently, the heme coordination state together with the energy barriers provided by the protein structure control the dynamics and affinity for NO-binding enzymes.
INSERM U696, Laboratoire d'Optique et Biosciences, Ecole Polytechnique, Palaiseau F91120, 91128 Palaiseau Cedex, France. michel.negrerie@polytechnique.fr
Michel
M
Negrerie
Sergei G
SG
Kruglik
Jean-Christophe
JC
Lambry
Marten H
MH
Vos
Jean-Louis
JL
Martin
Stefan
S
Franzen
D000818:Animals; D002417:Cattle; D006418:Heme / Q000737:chemistry*; D006801:Humans; D007093:Imidazoles / Q000737:chemistry; D007501:Iron / Q000737:chemistry*; D007700:Kinetics; D008024:Ligands; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation; D009211:Myoglobin / Q000737:chemistry / Q000235:genetics*; D009569:Nitric Oxide / Q000737:chemistry*; D009584:Nitrogen / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman; D013997:Time Factors
D007093:Imidazoles; D008024:Ligands; D009211:Myoglobin; D011506:Proteins; D009569:Nitric Oxide; D006418:Heme; C029899:imidazole; D007501:Iron; D009584:Nitrogen