Photochemical action spectrum

The existence of two separate but interacting photosystems in photosynthetic eukaryotes was demonstrated through analysis of the photochemical action spectrum of photosynthesis, in which the oxygen-evolving capacity as a function of light wavelength was determined (Figure 22.10). 

FIGURE 22.10 The photochemical action spectrum of photosynthesis. The qnantnm yield of photosynthesis as a fnnction of wavelength of incident light shows an abrnpt decrease above 680 nm, the so-called red drop. 

Knowing that carbon monoxide complexes of hemes are dissociated by light, Warburg and Negelein, in 1928, determined the photochemical action spectrum (see Chapter 23) for reversal of the carbon monoxide inhibition of respiration of the yeast Torula utilis. The spectrum closely resembled the absorption spectrum of known heme derivatives (Fig. 16-7). Thus, it was proposed that 02, as well as CO, combines with the iron of the heme group in the Atmungsferment. 

Carbon monoxide inhibited the 6/3-. la-, and 16a-hydroxylation of testosterone by rat liver microsomes to different extents. A C0/02 ratio of 0.5 inhibited the la-, 6/i-, and 16a-hydroxylation reactions by 14%, 25%, and 36%, respectively, and the ratio of C0/02 needed for 50% inhibition of testosterone hydroxylation in the 16a-, 6/3-, and 7a-positions was 0.93, 1.54, and 2.36, respectively (36,48). Studies on the photochemical action spectrum revealed that CO inhibition of the three hydroxylation reactions was maximally reversed by monochromatic light at 450 nm, but there were differences in the shape of the photochemical reactivation spectra for the 6/3-, la-, and 16a-hydroxylation reactions (36,48). The data from our laboratory summarized above and at the First International Symposium on Microsomes and Drug Oxidation in 1968 pointed to multiple cytochromes P450 with different catalytic activities that were under separate regulatory control (36,45,46), and we indicated that the actual number of cytochromes that participate in the multiple hydroxylation reactions must await the solubilization and purification of the microsomal system (36). The use of different inducers of liver microsomal monooxygenases caused selective increases in the concentration of specific cytochromes P450 in fiver microsomes that greatly facilitated the isolation and purification of these hemoproteins. 

Cooper DY, Levin S, Narasimhulu S, Rosenthal O, Estabrook RW. 1965. Photochemical action spectrum of the terminal oxidase of mixed function oxidase systems. Science 147 400-2... 

The adsorbate substrate complex excitation mechanism predicts an action spectrum analagous to the absorption spectra of the complex. The direct mechanism predicts a photochemical action spectrum similar to that of the gas phase molecule. The final mechanism, dissociative electron attachment (DEA), suggests that the action spectrum should be referenced to the absorbance of the substrate, modified by the surface work function and electron attachment cross section of the adsorbate. The DEA mechanism appears to be of importance for many metal and semiconductor substrates, especially for the case of photochemistry induced by anomalously low energy radiation. 

Recent studies of the photochemistry of Mo(CO)6 on Ag(l 11) 05 graphite 05 Cu(l 1 1 )70.71,106 and Si( 11 1 )70,71,106 have also shown clear evidence for direct adsorbate excitation. The main techniques employed were MS, EELS and TPD. That the electronic structure was essentially unperturbed on adsorption was confirmed by EELS.105 The photochemical action spectrum followed the gas phase spectrum rather closely, even to the extent of reproducing the charge transfer bands. This is very clear evidence for the direct excitation mechanism. There was an additional enhancement at 325 nm on Ag (111) due to the field enhancement associated with the resonance between the d bands and the Fermi level. Several interesting results were obtained with regard to the excited state quenching. Since the electronic transitions are... 

Conclusive evidence that a species of cytochrome P-450 was involved in the hydroxylation was presented by Okuda et al., who showed that the photochemical action spectrum for reversal of the carbon monoxide inhibition of 26-hydroxylation of 5)8-cholestane-3a,7a,12a-triol in rat liver exhibited a maximum at 450 nm [134]. Pedersen et al. [135] and Sato et al. [136] reported simultaneously that small amounts of cytochrome P-450 could be solubilized from the inner membranes of rat liver mitochondria that was active towards cholesterol as well as 5)8-cholestane-3a,7a,12a-triol in the presence of ferredoxin, ferredoxin reductase and NADPH. The mechanism of hydroxylation is thus the same as that operative in the biosynthesis of steroid hormones in the adrenals and in the la-hydroxylation of 25-hydroxyvitamin D in the kidney (Fig. 8). The liver mitochondrial cytochrome P-450 was not active in the presence of microsomal NADPH-cytochrome P-450 reductase [135,136]. Ferredoxin reductase as well as ferredoxin were active regardless of whether they were isolated from rat liver mitochondria or bovine adrenal mitochondria [133]. The partially purified cytochrome P-450 had a carbon monoxide difference spectrum similar to that of microsomal cytochrome P-450 from liver microsomes and adrenal mitochondria. In the work by Pedersen et al. [133], the concentration of mitochondrial cytochrome P-450 in rat liver mitochondria from untreated rats was calculated to be only about 0.1 nmole/mg protein. Treatment of rats with phenobarbital increased the specific content of cytochrome P-450 in the mitochondria more than 2-fold, without significant increase in the 26-hydroxylase activity. The carbon monoxide spectrum of the reduced cytochrome P-450 solubilized from liver mitochondria of phenobarbital-treated rats exhibited a spectral shift of about 2 nm as compared to the corresponding spectrum obtained in analysis of preparations from untreated rats. This was taken as evidence that more than one species of cytochrome P-450 was present in the preparation. It was later shown by Pedersen et al. [137] and Bjbrkhem et al. [138] that the preparation was also able to catalyse 25-hydroxylation of vitamin D3 and that different enzymes are involved in... 

If the degree of reversal of the CO inhibition by diflFerent monochromatic light bands of equal quantum intensity is plotted as function of wavelength, a photochemical action spectrum is obtained which agrees with the spectrophotometric difference spectrum of P-450 CO. Hydroxylase reactions for which the participation of cytochrome P-450 has been established by the action spectrum are listed in Table I. There is evidence that the hydroxylations of cholesterol by liver microsomes at position dp (rats) (32) and 7a (rabbits) (30) may also belong to this group of reactions. 

Photochemical Action Spectrum. The relative photochemical action spectrum of reconstituted ll)8-hydroxylase preparations is shown in Figure 3. The photochemical action of the individual monochromatic... 

Figure 3. Photochemical action spectrum of reconstituted llp-hydroxylase system. Curve is a composite of five experiments, three in the range 450-401 mfx and two in the range 450-500 mfx. Experimental procedure and composition of reaction mixture are described in Table 11 concentrations of Fp and NHI given in Flask 6 of Table II. Rate of corticosterone formation in the absence of CO averaged 9 nmole/mg. P-3 protein/min. Flasks were gassed with 4% O2-96% Ngor4% Og-4.2% CO-91.8% N/CO/O2 ratio = 1.05). The ordinate scale is relative light sensitivity Lx/L 50. L is calculated as described in Table III. The wavelength of monochromatic light of approximately equal intensity illuminating the reaction vessels is plotted on the abscissa...

In the following years cytochrome P450 was also found as an integral part of the 1 l 3-hydroxylase system in adrenal mitochondria Its involvement in the liver microsomal monooxygenation of codeine, acetanilide and cyclohexane was established using the technique of the photochemical action spectrum (Fig. 2). 

Fig. 2. Photochemical action spectrum of the reversibility of the carbon monoxide inhibited cyclohexane hydroxylation in rat liver microsomes. From )...

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