MbCO

X-ray crystallographic structures of myoglobin and hemoglobin were first completed in 19662 and 19753, respectively. Since then, many other X-ray crystallographic studies of deoxy- and oxy- as well as met-myoglobin and hemoglobin have been carried out.22,24 Additionally, researchers have studied the carbon monoxide bound moieties MbCO and HbCO as well as MbNO. Site-directed mutagenesis of residues near the active sites of Mb and Hb have yielded... 

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. 

A sensitive probe of electrostatic interactions in the distal pocket is provided by the structural and vibrational properties of the Fe-CO unit [9], The bound CO ligand exhibits three main infrared (IR) absorption bands, denoted Ao, A, and A3, with vibrational frequencies 1965 cm-1, 1949 cm, and 1933 cm, respectively. These bands, which change relative intensity and wave-number depending on temperature, pressure, pH, or solvent [10], are used to identify functionally different conformational substrates of MbCO, denoted taxonomic substates [11], Nevertheless the relationship between the A states and specific structural features of the protein has not yet been clarified. 

The A0 component is observed on reducing the pH or mutating the distal histidine residue (His64) [9a, 12]. In addition, an X-ray study of MbCO at low pH [3b] has demonstrated that His64 is far from the ligand and out of the heme pocket. On this basis the A0 state is usually associated with a protein substate in which the CO is in an apolar environment. 

In an attempt to aid interpretation of the IR spectrum of MbCO we decided to model the full protein by use of a hybrid quantum mechanics/molecular mechanics approach (QM/MM), to evaluate changes in the CO stretching frequency for different protein conformations. The QM/MM method used [44] combines a first-principles description of the active center with a force-field treatment (using the CHARMM force field) of the rest of the protein. The QM-MM boundary is modeled by use of link atoms (four in the heme vinyl and propionate substituents and one on the His64 residue). Our QM region will include the CO ligand, the porphyrin, and the axial imidazole (Fig. 3.13). The vinyl and propionate porphyrin substituents were not included, because we had previously found they did not affect the properties of the Fe-ligand bonds (Section 3.3.1). It was, on the other hand, crucial to include the imidazole of the proximal His (directly bonded to the... 

Classical simulations of MbCO using the CHARMM force field were performed for different tautomerization states of the distal histidine residue (His64) [33], These simulations showed that when His64 is protonated at N,5 (denoted the tautomer) it often rotates such that it exposes either the N,>—H bond or the un-protonated N atom to the CO, as depicted in Scheme 3.4. We... 

Pathway I was observed for all the 02 complexes studied, strained or unstrained, as well as for the unstrained CO-complexes. This particular pathway is the same one observed in the photodissociation of the natural heme complexes (3,4) (HbCO, MbCO, HbO and MbO ) with the exception that there is no detectable geminate recombination to the limit of our experiment, 50 ps. Pathway II, observed for the strained-CO complexes, reveals the presence of a fifth intermediate X found early in the dissociation that is either absent or undetectable in the natural or synthetic heme complexes following pathway I. The kinetics associated with the evolution of these intermediates will be discussed shortly. First, it is appropriate to examine in some detail the experimental AA difference spectra of two representative complexes, 1 -CO and 1-ET-CO. A discussion of 1-ST-CO and l-ET-O is also included for comparative purposes. ... 

Chance and co-workers have designed a flow system where the protein is continuously pumped optically using a tungsten or xenon flash lamp (764 nm). Using continuous illumination for various times and temperatures. Chance et al. have observed three intermediate states upon MbCO photolysis. At 40 K, a state with a recombination rate constant of 2 x 10 /s has been identified from two slower states with rate constants of 10 /s. 

Many methods of investigation of protein-ligand binding kinetics that are based on linear processes are of a pump-probe type. In this approach an optical pulse, called a pump, starts a photoreaction (such as dissociation of MbCO into Mb and CO), and its progress is probed a time At later. The probe could be, for example, a weak laser pulse, which detects the spectral changes in the heme during the protein-ligand recombination, or an x-ray pulse, which allows determination of the protein structure at a particular instant in time. 

In the next section, we describe two pump-probe approaches and review their use in experiments with MbCO. This places the nonlinear optical techniques of transient phase grating introduced in the subsequent section in their proper context. 

B. Overview of Transient Grating Results 1. MbCO in Aqueous Solutions... 

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