Myoglobin extension

There has been extensive work done on myoglobin, haemoglobin, Cytocln-ome-c, rhodopsin and bacteriorhodopsin. In fact, there are literally hundreds of articles on each of the above subjects. Flere we will consider haemoglobin [12]. The first tliree of these examples are based on the protohaeme unit, shown in figure Bl.2.10. 

Figure 26.5 (a) The o-helical secondary structure of proteins is stabilized by hydrogen bonds between the N—H group of one residue and the C=0 group four residues away, (b) The structure of myoglobin, a globular protein with extensive helical regions that are shown as coiled ribbons in this representation. 

Extensive myoglobin engineering work supports a major role for the distal His in stabilizing the oxy complex relative to the CO complex (182). O2 is a more polar molecule than CO and hence will interact more favorably with neighboring polar groups such as the distal His. This explanation is supported by theoretical work showing that even if CO does tilt or bend, the energetic cost is minimal (184), while the oxy... 

Meat discoloration studies typically involve a maximum of 5 days, with discoloration analy-sis being performed every day or on alternate days. The actual experimental time involved in the objective assessment of discoloration is not extensive and depends on the number of samples being analyzed. Colorimetric measurements with hand-held colorimeters are very rapid (three measurements per meat surface in < 1 min). Spectral scans of meat surfaces require 1 to 2 min. Extraction and analysis of ground meat products has the added step of homogenization and filtration prior to spectrophotometry, but relative to many laboratory procedures, this is relatively quick. Isolation and purification of preparative amounts of myoglobin requires only 2 to 3 days once appropriate preparations are made. Finally, metmyoglobin can be reduced to oxymyoglobin in 15 to 20 min. 

An obvious extension to 3D spectroscopy from 2D spectroscopy is the homonu-clear NOESY-NOESY [34]. There are two t variable times and one tj, which after Fourier transform provide three frequency domains. The 3D NOESY-NOESY spectrum of met-myoglobin cyanide, which contains low spin iron(III) in a heme moiety (see Fig. 5.7), has been successfully measured [35]. In Fig. 8.22 a slice of the 3D spectrum is shown at the I2-CH3 height. On the diagonal it shows all the dipolar connectivities between I2-CH3 and other protons off-diagonal... 

Fig. 4. Temperature dependence of the specific enthalpy of denaturation of myoglobin and ribonuclease A (per mole of amino acid residues) in solutions with pH and buffer providing maximal stability of these proteins and compensation of heat effects of ionization (see Privalov and Khechinashvili, 1974). The broken extension of the solid lines represents a region that is less certain due to uncertainty in the A°CP function (see Fig. 2). The dot-and-dash lines represent the functions calculated with the assumption that the denaturation heat capacity increment is temperature independent.

The replacement of native haem with metal-substituted porphyrin can be performed in two ways. In the first, iron ions are removed from the protein by treating native protein with anhydrous HF, followed by insertion of the appropriate metal to metal-free protein [57-59]. In the second, haem is removed either chemically or by recombination (preparing a proper recombinant protein), and then protein is reconstituted with metal-substituted porphyrins [60-64]. The Zn-substituted metal-loproteins such as cytochrome c [65-67], myoglobin [59,61, 62, 64, 68, 69], and haemoglobin [64, 68,70] have been extensively used to study photoinduced ET (PET) between modified proteins and their physiological redox partners. Interestingly, in haemoglobin with a and /3 subunits it was possible to determine ET parameters for... 

To provide an understanding of the importance of solvent mobility and the intrinsic protein energy surface, an MDS of proteins and surrounding solvent molecules at different temperatures has been performed. The simulation of myoglobin dynamics showed that solvent mobility is the dominant factor in determining protein atomic fluctuations above 180 K (Vitkup et ah, 2000). The drastic effects of water molecule dynamics on the intramolecular motion of RNase and xylase was demonstrated in recent computer simulation studies (Reat et al., 2000 Tarek et al, 2000). Extensive simulations were carried out to identify the time-scale of water attachment to lysozyme (Steprone et... 

Mossbauer spectra has been extensively used to probe the structure of the iron nucleus in biological FeIV=0 compounds. These include horseradish peroxidase compoundl[134,180,181], horseradish peroxidase compound II [182,183], horseradish peroxidase compound X [181], Japanese-radish peroxidase compounds I and II [184], chloroperoxidase compound I [185], cytochrome c peroxidase compound I [186] and ferryl myoglobin [183]. Examples of Mossbauer spectra attributed to non-porphyrin-bound FeIV are only available from synthetic model compounds. These include compounds with [130] and without [4-8] an FeIV=0 bond. 

Although both haemoglobin [155,231] and myoglobin produce ferryl iron and free radicals upon addition of H2O2, the latter reaction has been studied more extensively. In contrast to most peroxidases, at least two myoglobin-bound free radicals have been detected immediately after peroxide addition, both by room-temperature [142] and low-temperature EPR[137]. These decay rapidly and independently of the decay of the ferryl iron, which is stable for at least an hour. It is not necessary to add exogenous reductants to reduce the radicals it is possible that some of the electrons come from elsewhere on the protein as different, more stable, free radicals can be detected one hour subsequent to peroxide treatment [137,236]. 

One of the variables in the structures of the porphyrins present in heme proteins is the presence or absence of vinyl substituents on the periphery of the macrocycle. For example, b hemes have vinyl substituents whereas c hemes do not. Because of the sensitivity of such vinyl substituents during synthetic transformations, it has often been desirable to use octa-alkyl porphyrins in model studies of the spectroscopic properties of heme systems. The development of improved methods for the preparation of octa-alkyl porphyrins has likewise increased the availability of such porphyrins for model studies (20, 21). To assess the effect that replacement of the two vinyl substituents in protoporphyrin IX with alkyl (ethyl) groups has on the MCD properties of the heme system, an extensive and systematic study of the MCD properties of mesoheme IX-reconstituted myoglobin and horseradish peroxidase in comparison with the spectra of the native protoheme-bound proteins has been carried out (22). The structures of these two porphyrins are shown in Figure 3. 

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