Histidine proximal

Even though the iron atoms are separated in haemoglobin by about 25 A, communication between them is still able to occur and this has been postulated to involve a trigger mechanism (Perutz, 1971). The trigger is the movement of the proximal histidine as dioxygen binds to (or is released from) the Fe(n) and results in interconversion between the T and R structures. This movement causes a conformational change which is transmitted through the protein to the other iron sites. X-ray studies indicate that relative shifts of up to 6 A at subunit interfaces occur between the T and R states (Perutz, 1978). 

Nanosecond time-resolved crystallography of MbCO has been discussed in Section 3.7.2.3 of Chapter 3.46 After firing a 10-ns burst of laser light to break the CO-Fe bond, these researchers produced a diffraction image of the crystal through application of a 150-ps X-ray pulse. They are able to show release of the CO molecule, displacement of the Fe ion toward the proximal histidine, and recombination of the dissociated CO by about 100 ps. Essentially their results compare well with other spectroscopic studies of HbCO, MbCO and their models. 

Figure 3.4 shows the models used in the calculations of the active center. They are of the type FeP-AB (FeP = iron-porphyrin, AB = CO, NO, 02), Heme-AB (AB = CO, NO, 02), and FeP(Im)-AB (Im=imidazole). The axial imidazole ligand mimics the effect of the proximal histidine amino acid (Fig. 3.2). 

Activation parameters for the reaction of NO with metMb, Eq. (15), were determined in this laboratory and in collaboration with van Eldik and Stochel (Table II) (23). Comparison of these activation parameters with those determined for reactions of NO with the water soluble ferri-heme complexes Fem(TPPS)(H20)2 and Feni(TMPS)(H20)2 (Table II) demonstrate that the latter compounds represent reasonable models for the kinetics for the analogous reaction with metMb. For example, the kon step would appear to be defined largely by the H20 lability of metMb(H20), although it is clear that the diffusion through protein channels, the distal residues and the proximal histidine binding to the Fe(III) center must all influence the NO binding kinetics (23,24). These properties may indeed be reflected in the lower AS values for both the on and off reactions on metMb. In a related study, Cao et al. recently... 

Measurements of the proximal histidine-iron stretching frequency by Resonance Raman spectroscopy revealed that this bond is very weak in relation to other heme protein systems (vFe.His = 204 cm-1) (130). Formation of the sGC-NO complex labilizes this ligand resulting in the formation of a 5-coordinate high spin iron(II) complex, and the conformational change responsible for the several hundred-fold increase in catalytic activity (126,129,130). 

The haem is tightly bound to the protein in a hydrophobic pocket formed principally by helices E and F and by a single coordinate bond between the imidazole of His F8, termed the proximal histidine (Figure 13.6), and the ferrous iron, which is some 0.6 A out of the plane of the domed porphyrin ring. A second His residue, His E7 (the distal histidine), is too far away from the iron atom to coordinate with it in the deoxy state. 

The proximal calcium binding site is coupled to the heme group by virtue of the fact that one of its ligands, Thrl71, is adjacent to the proximal histidine residue, Hisl70 (Fig. 4). The results of site-directed mutagenesis studies at this position are awaited with interest. An illustration of the importance of both calcium sites to the structure and function of HRP C is afforded by the need to incorporate calcium as a component of in vitro folding mixtures to obtain active recombinant enzyme from solubilized inclusion bodies (64). 

The optical spectral data for the NP2 -NO and NP3 -NO complexes at pH 5.5 suggest that there may be a difference in Fe-N bond strength of the proximal histidine ligand for these two proteins that is only apparent when the metal is reduced to Fe(II). Thus, the kinetic differences in NO release behavior of the NP1,4 group and the NP2,3 group, discussed later, could in part be related to differences in heme-histidine bond strength. If so, then it must be concluded that a weakened Fe-N bond strength produces a more stable Fe-NO complex, since the Ki values for NP2,3-NO are much smaller than for NPl,4-NO (50) (see Section IV). 

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