Heme chromophore

Figure B3.5.5 Near-UV CD spectra. (A) Bovine a -casein peptide under a variety of conditions (data from Alaimo et al., 1999). Peptide concentration 0.631 mg/ml in 2 mM PIPES, 4 mM KCI, pH 6.75 scan rate 40 sec/nm path length 10 mm bandwidth 1.5 nm. The loss of aromatic dichroism with increasing temperature indicates denaturation, which is, however, not complete at 70°C or in 6 M guanidine hydrochloride. The shift in maximum wavelength indicates loss of tryptophan asymmetry, but less so of tyrosine. (B) Seed coat soybean peroxidase under native and denaturing conditions (data from Kamal and Behere, 2002). Protein concentration 15 pM and path length 10 mm. The negative aromatic band centered around 280 nm and the Soret band around 410 nm both disappear at 90°C, indicating the loss of net conformational asymmetry of the aromatic and heme chromophores.

Chloramphenicol and secobarbital exhibit properties similar to those of tienilic acid, but they have not been studied in humans (11). Oxidative dechlorination of chloramphenicol with formation of reactive acyl chlorides appears to be an important metabolic pathway for irreversible inhibition of CYP. Chloramphenicol binds to CYP, and subsequent substrate hydroxylation and product release are not impaired. The inhibition of CYP oxidation and the inhibition of endogenous NADPH oxidase activity suggest that some modification of the CYP has taken place, which inhibits its ability to accept electrons from the CYP reductase (11). Secobarbital completely inactivates rat CYP2B1 functionally, with partial loss of the heme chromophore. Isolation of the N-alkylated secobarbital heme adduct and the modified CYP2B1 protein revealed that the metabolite partitioned between heme N-alkylation, CYP2B1 protein modification, and epoxidation. A small fraction of the prosthetic heme modifies the protein and contributes to the CYP2B1 inactivation (12). 

Figure 8. Absorption spectrum of ferriheme undecapeptide (—) and of ferroheme undecapeptide (hemochromogen) (- ) in 0.05 M sodium tetraborate, pH 9. The right-hand ordinate is for the Soret (y) band and for the absorbances at wavelengths shorter than 210 nm. The hemochromogen bands are labelled a-s. As there are no residues in the undecapeptide with chromophores absorbing at wavelengths greater than 240 nm, the 280 nm, e and S bands are clearly due to the heme chromophore. Reproduced, with permission, from [11],...

Furalylline (Figure 7.24), a potent selective inhibitor of human CYP1A2, causes time- and NADPH-dependent inactivation of this enz-yme368, 369 CYPIAI, -2A6, -2B6, -2C9, -2C19, -2D6, -3A4, and -2E1 are not similarly inactivated, although the evidence suggests that other enzymes can be inhibited . Clinical studies confirm that fiirafylline can almost completely suppress in vivo human CYPl A2 function This loss of function is paralleled by a similar loss of the heme chromophore, with — 23 pM and — 0.87 min , and a partition ratio of 3-6 substrate... 

Using biomolecules with chromophoric groups the Raman bands are both resonance (RRS) — and surface enhanced (RRS + SERS = SERRS). Instead of the usual term SERS, the Raman effect is, in this case, called surface enhanced resonance Raman scattering (SERRS). SERRS spectroscopy was first applied to biochemistry of heme chromophores by Cotton et al. Since then, SERS and SERRS have been extended to systematic investigations of biomolecules in the adsorbed state... 

As discussed before in the case of nucleic acids the authors have also considered the incidence of the interfacial conformation of the hemoproteins on the appearance of the SERRS signals from the chromophores. Although under their Raman conditions no protein vibration can be observed, the possibility of heme loss or protein denatura-tion are envisaged to explain a direct interaction of the heme chromophores with the electrode surface in the case of the adsorl Mb. extensive denaturation of Cytc at the electrode appears unlikely to the authors on the basis of the close correspondence of the surface and solution spectra. Furthermore, the sluggish electron transfer kinetics measured by cyclic voltammetry in the case of Cytc is also an argument in favour of some structural hindrance for the accessibility to the heme chromophore in the adsorbed state of Cytc. This electrochemical aspect of the behaviour of Cytc has very recently incited Cotton et al. and Tanigushi et al. to modify the silver and gold electrode surface in order to accelerate the electron transfer. The authors show that in the presence of 4,4-bipyridine bis (4-pyridyl)disulfide and purine an enhancement of the quasi-reversible redox process is possible. The SERRS spectroscopy has also permitted the characterization of the surface of the modified silver electrode. It has teen thus shown, that in presence of both pyridine derivates the direct adsorption of the heme chromophore is not detected while in presence of purine a coadsorption of Cytc and purine occurs In the case of the Ag-bipyridyl modified electrode the cyclicvoltammetric and SERRS data indicate that the bipyridyl forms an Ag(I) complex on Ag electrodes with the appropriate redox potential to mediate electron transfer between the electrode and cytochrome c. 

In the case of cytochrome cdi (Cyt cdi) found in many facultative, anaerobic denitrifying bacteria, the SERRS spectroscopy has been used to obtain preliminary heme structure/environment comparisons between cyt cdx from two bacteria sources. Pseudomonas and Paracoccus. The difficulty encountered in preparing sufficient quantities of cyt cdi, can be thus solved by the low product consuming SERRS method. By exciting selectively with the 514.5 nm and 460 nm lines of an Ar laser, it is possible to produce successively an enhancement of RR scattering of reduced heme c, see Fig. 34, and heme di, see Fig. 35, in the protein complexes (3 x 10 M) adsorbed on Ag at —0.6 V vs. SCE. A comparison between the SERRS spectra of cdi proteins from the bacteria suggests differences in the heme chromophore and peripheral substituents. 

A variety of compounds containing an olefinic bond, such as ethylene, allylisopropylacetamide (AlA), and secobaibital, can form covalent adducts on the nitrogen of the porphyrin group of the prosthetic heme leading to inactivation [382-385], Secobaibital has been shown to completely inactivate CYP2B1 with only partial loss of the heme chromophore [384, 386, 387], Isolation of the modified CYP2B1 protein and the A -alkylated porphyrins indicates that the reactive compound partitions between protein modifiea-tion, A -alkylation of the heme, and formation of an epoxide metabolite in the ratio of 0,2 0.8 59, respectively [387] (Fig, 5.17), The formation of a heme adduct in the active site of CYP2B1 was confirmed spectrally based on its typical absorption maximum at 445 nm, a characteristic fea-... 

An illustration of the spectroscopic emergence of a strong isolated chromophore within the interior of a substantially large protein is given by this group s spectroscopy of the cytochrome-c system. The Soret band near 410 nm is nicely observed in the [M-6H] electron photodetachment action spectrum for cytochrome-c, containing the Fe(lll) heme chromophore [136] (Fig. 14). The gas-phase peak is very close to the position observed in solution, suggesting that the chromophore is well shielded from solvent interactions in solution. 

Type I substrates such as hexobarbital, ethylmorphine and testosterone which cause loss of absorbance at 420nm with a concomitant increase at 385nm, and Type II substrates such as aniline, pyridine and acetanilide which cause an increase in absorbance at 430nm and a decrease at 400nm. Such alterations in the heme chromophore reflect interaction at or near the active site of the enzyme. Type II substrates have nonbonding electron pairs which are thought to interact with the heme iron. Three types of EPR spectra have been seen for oxidized cytochrome P-450, two low spin forms and one high spin form. One of the low spin forms is associated with substrate-free oxidized P-450, while the other is observed... 

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