Heme Environment

As in the other members of the superfamily, the heme pocket of ligninolytic peroxidases includes two conserved histidine residues disposed above and below the heme plane (Fig. 3.4a). The second histidine acts as the fifth ligand of the heme iron, occupying a proximal position, while the first one is at a higher distance being, therefore, called distal histidine (by extension, the regions located below and above the heme plane are also called proximal and distal regions). 

Four more amino acid residues are conserved at the distal (arginine and phenylalanine) and proximal (aspartate and phenylalanine) sides of the heme pocket in all structurally characterized ligninolytic peroxidases [33], two of them (distal arginine and proximal aspartate) also being conserved in the other members of the superfamily. 

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A model for the allosteric behavior of hemoglobin is based on recent observations that oxygen is accessible only to the heme groups of the a-chains when hemoglobin is in the T conformational state. Perutz has pointed out that the heme environment of /3-chains in the T state is virtually inaccessible because of steric hindrance by amino acid residues in the E helix. This hindrance dis-... 

Pig. 2. Comparison of the heme environments at the active sites of horse heart myoglobin 40) and yeast cytochrome c peroxidase 41). 

Perhaps the most fundamental fimctional property of a heme prosthetic group at the active site of a heme protein is the relative stability of the reduced and oxidized states of the heme iron. A number of structural characteristics of the heme binding environment provided by the apo-protein have been identified as contributing to the regulation of this equilibrium and have been reviewed elsewhere 82-84). Although a comprehensive discussion of these factors is not possible in the space available here, they can be summarized briefly. The two most significant influences of the reduction potential of the heme iron appear to be the dielectric constant of the heme environment 81, 83) and the chemical... 

Fig. 9. EPR spectra of heme-hemopexin and heme-N-domain. X-band EPR spectra at 4 K of ferri-mesoheme-hemopexin (a) and ferri-mesoheme-N-domain (b) are shown. The concentration of both heme complexes was 0.15 mM in 50 50 (v/v) 10 mM sodium phosphate/150 mM NaCl (pH 7.2) glycerol. The g-value scale is noted at the top and the -values observed are noted in each spectrum. Although both complexes are low-spin (some adventitious high-spin iron is present), the differences in g-values indicate nonidentical heme environments in the two complexes (.114).

Fig. 10. H NMR spectra of rabbit hemopexin and domain. The 400 MHz H NMR spectra at 298 K of rabbit apo- and ferri-mesoheme-hemopexin are shown in panels A and B, and the spectra of apo- and mesoheme-N-domain in panels C and D, respectively. The spectra demonstrate that the heme environment in the intact protein is distinct from that in the N-domain. The heme resonances are broadened in the N-domain, consistent with a greater accessibility to solvent 114).

The ferro-complex CD spectrum shows that reduction of the heme iron alters the heme environment. Redox-induced protein conformation changes could alter the S5unmetry in the heme pocket or produce two binding modes for the reduced complex whose asymmetries nearly cancel each other. Redox-linked conformational changes are especially interesting in view of recent findings of oxido-reductase activity associated with the heme-hemopexin-receptor interaction (89). 

Monomeric hemes possess a mirror plane and are hence achiral (151). Incorporation of the heme macrocycle into the anisotropic protein matrix distorts the heme environment, inducing a circular dichroism spectrum (57, 152, 153). From the design standpoint, the presence of an induced heme CD spectrum qualitatively confirms intimate communication between the heme and the surrounding protein matrix, which indicates the heme is most likely specifically bound. This spectroscopic signature serves as a first indication that the heme resides within the designed protein scaffold and has been used by various groups to... 

Hg. 19j The myoglobin molecule (a) the folding of Ihe polypeptide chain about the heme group (represented by the disk) (b) close-up view of the heme environment. (Modified from Kendrew. J. C. Dickerson. R. E.. Strand berg, B. E.. Hart. R. C. Davies. D. R. Phillips D. C.. Shore. V. C. Nature 1960.185,422-423. Reproduced with permission.)... 

Fig. 19.7 Stercoview of superimposed heme environments in oxyhemoglobin and oxymyoglobin. Solid lines denote HbO and dashed lines MbOj. Note the difference in the Fe—01—D2 bond angles and the presumed hydrogen bond (dotted line) to the histidine (His E7). [From Shaanan, B. Nature (LonJvn) 982,296, 683. Reproduced with permission.]...

Another property that distinguishes various cytochromes is the redox potential E ° (Table 6-8), which in this discussion is given for pH 7.0. Cytochromes carry electrons between other oxidoreductase proteins of widely varying values of E°. Because of the various heme environments cytochromes have greatly differing values of E°, allowing them to function in many different biochemical systems. 97a/97b For mitochondrial cytochrome c the value of E ° is + 0.265 V but for the closely related cytochrome/of chloroplasts it is +0.365 V and for cytochrome c3 of Desulfovibrio about -0.330 V. There is more than an 0.6-volt difference between E ° ... 

New insights into the peroxide binding and in the catalysis, obtained through site-directed mutagenesis, have led to much-improved understanding of heme peroxidases and their possible applications. New crystal structures of peroxidases have provided much more information on the local heme environments [32],... 

C Resonances from Heme Environments 1. Heme Pockets in Unligated Form... 

Compared with plant and other peroxidases, white-rot fungal peroxidases are characterized by their high redox potential, related to the architecture of the heme environment (see Chap. 4). This is required to perform their role in nature, namely the oxidative biodegradation of the recalcitrant lignin polymer present in the cell wall of all vascular plants [30-32], By contrast, one of the roles of plant peroxidases... 

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