Abstract— The triplet state characteristics (spectrum, lifetime and quantum yield) for four dye sensi tisers [methylene blue (MB), erythrosin (ER), haematoporphyrin (HP) and riboflavin (RF)] were determined in methanol by laser flash photolysis and singlet oxygen yields (0.60 to 0.48) from time‐resolved measurements of the 1270 nm near infrared emission. The reaction of singlet oxygen with four long chain unsaturated phenyl esters [oleate (18: 1), linoleate (18: 2), linolenate (18: 3) and arachidonate (20: 4)] was followed quantitatively using the singlet oxygen luminescence technique and also, after continuous420–700 nm irradiation, by HPLC and other analysis of the isomeric product monohydroperoxides. The overall quantum yield of photooxidation (∼10‐2) was shown to be consistent with the observed singlet oxygen quenching constants(2–12 times 104 dm3 mol‐1 s‐1) for the four esters studied and the singlet oxygen lifetime in methanol (τ∼ 9 μs). The isomer product distribution was interpreted in terms of a dual singlet oxygen and radical mechanism, the radical contribution increasing with sensitiser in the order ER = MB < HP ≪ RF, but also showing some dependence on substrate unsaturation. Evidence is presented for singlet oxygen quenching by MB and RF (kO= 1.6 and 6.0 times 107 dm3 mol‐1 s‐1) and for the accelerated photobleaching of the dye sensitisers in the presence of the unsaturated esters.

Laser flash photolysis has been used to characterise the triplet state of metal-free (H2Pc) and copper phthalocyanine (CuPc) in dilute (lo-" mol dm-3) solution in chloronaphthalene. This first observation of the triplet state of CuPc indicates that its lifetime is very short (-35ns), but the quantum efficiency of triplet formation (>0.70) is greater than that for HzPc (4.14). The triplet absorption maximum for both compounds occur at ~4 8 0 nm, but the triplet energy level of CuPc (150 kJ) is higher than that for H2Pc (120 kJ).More concentrated solutions (10-4-10-5 mol dm-3) of both phthalocyanines give short-lived (100 ns) and long-lived (2.5 ps) transients, which have been attributed to the formation of excitons in microcrystalline particles dispersed in the chloronaphthalene.

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Flavocytochrome b2 from Saccharomyces cerevisiae is an l-lactate dehydrogenase which exhibits only barely detectable activity levels towards another 2-hydroxyacid, l-mandelate. Using protein engineering methods we have altered the active site of flavocytochrome b2 and successfully introduced substantial mandelate dehydrogenase activity into the enzyme. Changes to Ala-198 and Leu-230 have significant effects on the ability of the enzyme to utilize l-mandelate as a substrate. The double mutation of Ala-198-->Gly and Leu-230-->Ala results in an enzyme with a kcat value (25 degrees C) with L-mandelate of 8.5 s-1, which represents an increase of greater than 400-fold over the wild-type enzyme. Perhaps more significantly, the mutant enzyme has a catalytic efficiency (as judged by kcat/Km values) that is 6-fold higher with l-mandelate than it is with L-lactate. Closer examination of the X-ray structure of S. cerevisiae flavocytochrome b2 led us to conclude that one of the haem propionate groups might interfere with the binding of L-mandelate at the active site of the enzyme. To test this idea, the activity with l-mandelate of the independently expressed flavodehydrogenase domain (FDH), was examined and found to be higher than that seen with the wild-type enzyme. In addition, the double mutation of Ala-198-->Gly and Leu-230-->Ala introduced into FDH produced the greatest mandelate dehydrogenase activity increase, with a kcat value more than 700-fold greater than that seen with the wild-type holoenzyme. In addition, the enzyme efficiency (kcat/Km) of this mutant enzyme was more than 20-fold greater with L-mandelate than with l-lactate. We have therefore succeeded in constructing an enzyme which is now a better mandelate dehydrogenase than a lactate dehydrogenase.