Porphyrins porphyrin-enzyme complex

There are two possible modes of dissociation of the porphyrin from the enzyme active site. If solution samples are used, the dissociated porphyrin is blotted or rinsed away from the surface with the excess analyte solution. In this case, the absorbance intensity due to the porphyrin-enzyme complex is reduced in the waveguide spectrum by the percentage of complexes impacted (Figure 12.5, Panel D). The difference spectrum calculated as pre-exposure minus postexposure shows a loss in absorbance only at the characteristic peak for that particular porphyrin-enzyme interaction (Figure 12.6). Thus, it is possible to discriminate the binding of analyte(s) to different enzymes co-immobilized on a mixed bed surface containing different enzyme-porphyrin complexes. 

Several porphyrins bind OPH with nniqne spectrophotometric characteristics resnlting however, in order to form a porphyrin-enzyme complex that is sensitive to the presence of snbstrates of the enzyme, a copper-complexed porphyrin is necessary [30]. Two candidates, a copper-complexed TPPSi and copper-complexed TPPCi (mono(4-carboxy phenyl) porphyrin Rj = CO2A R2 = 803 ) inhibit the activity of OPH in a mixed manner. Mixed inhibition is the inhibition of enzyme activity in a manner such that the maximal enzymatic rate and the concentration needed to achieve half of that rate are both changed. The intersection of the cnrves in the absence and presence of the inhibitor occnrs in the second qnadrant of the Lineweaver-Bnrk plot. Mixed type inhibition involves the interaction of the inhibitor at two or more locations on the enzyme with one of these being the active site. The spectrophotometric characteristics of the porphyrin-enzyme complex are different depending on whether the apo or wild-type enzyme is bonnd by the copper-complexed porphyrins however, the spectrophotometric characteristics are identical for the interaction of TPPSi or TPPCi with either version of the enzyme. Other porphyrins snch as zinc-and iron-complexed TPPSi as well as the metal-free TPPSi and TPPCi do not inhibit the enzymatic activity of OPH. 

The systems described thns far are capable only of discrimination between substrates or inhibitors and those componnds that do not bind the enzyme active site. The hydrolysis of componnds snch as conmaphos and paraoxon prodnces products that absorb in the visible spectrnm. If a fixed path length standing drop were used for sample application (described earlier), the absorbance increase at wavelengths indicative of these prodncts together with the decrease in the absorbance of the characteristic peak of the porphyrin-enzyme complex would allow further identification of the analyte detected. 

As a first step in this direction, a surface consisting of electric eel AChE and horse serum BChE was designed. Both of these enzymes are inhibited by TPPSi and the characteristic absorbance peaks for the porphyrin-enzyme complexes are different (421 vs. 446 nm). This allows for co-immobilization of the two enzymes from a simple mixture of equal concentrations onto the entire slide surface [25]. Exposure to those compounds inhibiting BChE competitively results in a loss in absorbance at 421 nm while compounds inhibiting AChE competitively result in a loss at 446 nm. Compounds inhibiting both enzymes result in a loss at both 421 nm and 446 nm. This combination of two enzymes allows for class discrimination of those compounds, which are inhibitors of BChE, inhibitors of AChE, inhibitors of both enzymes, and inhibitors of neither enzyme. Detection limits are approximately the same for the dual enzyme system as those observed for the single enzyme systems. 

Figure 1. The interaction of analyte with the porphyrin-enzyme complex results in changes in the porphyrin absorbance spectrum.

The reversible inhibition of an enzyme by an association-sensitive colorimetric agent is the novel aspect of this detection technique. In detail, the poiphyrin interacts with the enzyme in a manner that requires its dissociation to allow analyte binding. The porphyrin-enzyme complex absorbance spectrum is different from that of the porphyrin alone. Changes in the absorbance spectrum that occur upon dissociation of the porphyrin from the enzyme are used to indicate the presence of the analyte. In addition, the degree of change is analyte concentration dependent. 

N—Fe(IV)Por complexes. Oxo iron(IV) porphyrin cation radical complexes, [O—Fe(IV)Por ], are important intermediates in oxygen atom transfer reactions. Compound I of the enzymes catalase and peroxidase have this formulation, as does the active intermediate in the catalytic cycle of cytochrome P Q. Similar intermediates are invoked in the extensively investigated hydroxylations and epoxidations of hydrocarbon substrates cataly2ed by iron porphyrins in the presence of such oxidizing agents as iodosylbenzene, NaOCl, peroxides, and air. 

The first test case was the ferrous high-spin state (Fe, S = 2) in the picket-fence porphyrin acetate complex [Fe(CH3COO)(TPpivP)] [13, 23], which is a model for the prosthetic group termed P460 of the multiheme enzyme hydroxyl-amine oxidoreductase from the bacterium Nitrosomonas europeae. Both the picket-fence porphyrin and the protein P460 exhibit an extraordinarily large quadrupole splitting, as observed by conventional Mossbauer studies [56]. 

The entire iron-porphyrin-protein complex is called a cytochrome and such proteins are important electron-transfer components of cells. Generally, access to the macromolecular region in which the oxidation reactions occur is via a hydrophobic channel through the protein (Mueller et al., 1995). As a result, organic substrates are transferred from aqueous solution into the enzyme s active site primarily due to their hydrophobicity and are limited by their size. This important feature seems very appropriate hydrophobic molecules are selected to associate with this enzyme, and these are precisely the ones that are most difficult for organisms to avoid accumulating from a surrounding aquatic environment. 

Rapid development of this area followed the discovery of routes to these complexes, either by ready conversion of terminal alkynes to vinylidene complexes in reactions with manganese, rhenium, and the iron-group metal complexes (11-14) or by protonation or alkylation of some metal Recent work has demonstrated the importance of vinylidene complexes in the metabolism of some chlorinated hydrocarbons (DDT) using iron porphyrin-based enzymes (15). Interconversions of alkyne and vinylidene ligands occur readily on multimetal centers. Several reactions involving organometallic reagents may proceed via intermediate vinylidene complexes. 

This chapter is divided into two sections. Section 6.1 is concerned with applications of Raman spectroscopy to biochemistry. Related topics to this section are found in Section 3.3.3 of Chapter 3 (SER spectra of dipeptides) and Section 4.1.2 of Chapter 4 (Raman (RR) spectra of peptides, proteins, porphyrins, enzymes and nucleic acids), Section 6.2 describes medical applications of Raman spectroscopy as analytical and diagnostic tools. In contrast to biochemical samples discussed in the former section, medical samples in the latter section contain a number of components such as proteins, nucleic acids, carbohydrates and lipids, etc. Thus, Raman spectra of medical samples are much more complex and must be interpreted with caution. 

In the early 1970s it was discovered that P-450 cytochromes are irreversibly inhibited during the metabolism of xenobiotics (1). The formation of a modified heme prosthetic group is associated with enzyme inhibition and subsequent studies have identified these modified complexes as N-alkylated protoporphyrin-IX (2). The chemistry of N-sub-stituted porphyrins was comprehensively reviewed by Lavallee in 1987 (3). Since that time, there have been many significant contributions to this field by several groups. The goal of this chapter is to summarize some of this work as it relates to the mechanism of formation and reactivity of iron N-alkyl porphyrins. Biomimetic model complexes have played an important role in elucidating the chemistry of N-alkyl hemes in much the same way that synthetic iron tetraarylporphyrins have aided... 

Cytochrome c oxidase is the terminal member of the respiratory chain in all animals and plants, aerobic yeasts, and some bacteria." " This enzyme is always found associated with a membrane the inner mitochondrial membrane in higher organisms or the cell membrane in bacteria. It is a large, complex, multisubunit enzyme whose characterization has been complicated by its size, by the fact that it is membrane-bound, and by the diversity of the four redox metal sites, i.e., two copper ions and two heme iron units, each of which is found in a different type of environment within the protein. Because of the complexity of this system and the absence of detailed structural information, spectroscopic studies of this enzyme and comparisons of spectral properties with 02-binding proteins (see Chapter 4) and with model iron-porphyrin and copper complexes have been invaluable in its characterization. 

In this case, a ferric porphyrin peroxo complex plays the role of the analogue of the reactive intermediate implicated in the third oxidative step of aromatase, the enzyme responsible for the conversion of androgens to estrogens (Scheme XI. 16). 

Cytochromes are porphyrin iron complexes bound to proteins which, as enzymes, play an important role in the oxidative phosphorylation in the respiratory cycle. 

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