Role of Heme Iron Coordination and Protein Structure in the Dynamics and Geminate Rebinding of Nitric Oxide to the H93G Myoglobin Mutant

Abstract

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 timeresolved 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 (ϳ10 and ϳ100 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.Nitric oxide acts as a signal transmitter in several physiological pathways (1-3) because it readily diffuses through cell membranes and reacts with the heme iron. It has been proposed that the ligation of NO 2 to myoglobin has a physiological role for the in vivo regulation of the NO concentration in muscle cells (4). Numerous hemoproteins are involved in diatomic ligand binding, such as CO, O 2 , and NO, and their regulation closely depends upon the dynamic behavior of these ligands. We have shown that the dynamics of nitric oxide, and hence the geminate rebinding to the heme, are closely related to the protein function and subtly controlled by the protein structure. This control is evident when comparing endothelial NO synthase (5) and soluble guanylate cyclase (6), which are the source and the receptor of NO in cells, respectively, in which NO acts as a second messenger for signal transduction. These two proteins display a very contrasting behavior with respect to geminate rebinding of NO, which is multiphasic and relatively slow for endothelial nitric-oxide synthase, including important nanosecond phases correlated with the release of newly synthesized NO. On the other hand, NO recombination is mono-exponential and ultrafast for sGC, revealin...

Molecular Basis for Nitric Oxide Dynamics and Affinity with Alcaligenes xylosoxidans Cytochrome c´

“…Remarkably, the rebinding of NO to the heme alone in solution is also mono-exponential (7.5 ps) but with a constant base line (8%) that corresponds to NO escape into the solvent (46,47). Thus, a time constant of ϳ7 ps for NO rebinding to the 4-coordinate heme after dissociation (either thermal or photoinduced) seems to be the lower limit for this barrierless process when no protein structure is involved, in agreement with calculations (16,48) and with temperature-dependent rebinding measurements (42).…”

“…It is noteworthy that, despite the efficient and fast geminate recombination, the k off value for 5c-NO AXCP (4.1 ϫ 10 Ϫ4 s Ϫ1 ) is of the same order of magnitude as 5c-NO heme protein complexes such as sGC ( (16). The high geminate recombination efficiency imposed by the proximal heme environment of AXCP implies that the typical k off value is due to an elevated value of k 1 .…”

“…This so-called trans effect is due to a weakening of the axial Fe-His bond caused by the trans-coordination of NO and is governed by protein structure (15). In 5c-NO species, the probability of fast NO rebinding after dissociation (either thermal or photo-induced) is increased (16) and tends to a single phase as observed in sGC (10). In sGC, the breaking of the proximal Fe-His bond in the resting state of the enzyme triggers the catalytic activity for cGMP production, whereas in AXCP the role of the proximal bond breaking and formation of a 5c-NO complex in a second order reaction (17,18) is not known.…”

Dynamics of Methionine Ligand Rebinding in Cytochrome c

“…Met80 rebinding involves at least two electronic states: 1,4,11,20 the ground state singlet (s), associated with the bonded state, and the quintet (q), associated with the unbonded state. In this work, the classical CHARMM force field in the heme pocket has been extended by incorporating ab initio PES calculations.…”

Analysis of Mechanism-Based Inhibition of CYP 3A4 by a Series of Fluoroquinolone Antibacterial Agents

“…Therefore, the amine nitrogen and heme iron cannot keep the optimal distance and the angle (C-N-Fe) to form an MI complex. A previous study reported that the potential energy of coordinate binding between the heme iron of myoglobin and nitric oxide depends on the distance (Negrerie et al, 2006). The potential energy reached the maximum at a distance of approximately 1.8 Å and then decreased dramatically according to dissociation between the heme iron and nitric oxide.…”