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CYP119 from Sulfolobus solfataricus is the first well-characterized thermophilic cytochrome P450 enzyme. The endogenous substrate for this enzyme is not known but it hydroxylates lauric acid in a reaction supported by surrogate mesophilic electron donors. However, reconstitution of a high-temperature catalytic system requires identification of the normal thermophilic electron donor partners of CYP119. Here, we describe cloning, expression in Escherichia coli, and characterization of the requisite electron donor partners from S. solfataricus. One is a thermostable ferredoxin and the second a 2-oxoacid-ferredoxin oxidoreductase that utilizes pyruvic acid rather than NAD(P)H as the source of reducing equivalents. CYP119 is the only cytochrome P450 to date known to obtain electrons from a non-NAD(P)H-dependent protein. The two thermophilic partners have been used to reconstitute a catalytic system that hydroxylates lauric acid at 70 degrees C, and the optimal conditions for this system have been defined. This first high-temperature in vitro catalytic system represents an important step in the development of industrially relevant catalysts.

Two notable features of the thermophilic CYP119, an Arg154-Glu212 salt bridge between the F-G loop and the I helix and an extended aromatic cluster, were studied to determine their contributions to the thermal stability of the enzyme. Site-specific mutants of the salt bridge (Arg154, Glu212) and aromatic cluster (Tyr2, Trp4, Trp231, Tyr250, Trp281) were expressed and purified. The substrate-binding and kinetic constants for lauric acid hydroxylation are little affected in most mutants, but the E212D mutant is inactive and the R154Q mutant has higher K(s),K(m), and k(cat) values. The salt bridge mutants, like wild-type CYP119, melt at 91+/-1 degrees C, whereas mutation of individual residues in the extended aromatic cluster lowers the T(m) by 10-15 degrees C even though no change is observed on mutation of an unrelated aromatic residue. The extended aromatic cluster, but not the Arg154-Glu212 salt bridge, contributes to the thermal stability of CYP119.

The cytochrome P450 superfamily of enzymes catalyzes a broad range of oxidative processes involved in the metabolism of fatty acids, biosynthesis of sterols, and elimination of drugs and xenobiotics. Application of the unique properties of P450 enzymes as fine biocatalysts in biotechnology is limited due to their thermal instability and the requirement for auxiliary electron-donor proteins and cofactors. CYP119, a thermophilic P450 enzyme from Sulfolobus solfataricus, was characterized some time ago, but no high-temperature redox partners have been available for it. Here we report reconstitution of CYP119 with a novel high-temperature electron-donor system consisting of a ferredoxin and 2-oxoacid:ferredoxin oxidoreductase from Sulfolobus tokodaii strain 7 that, unlike all other known P450 electron-donor partners, utilizes coenzyme-A and pyruvic acid rather than NADH or NADPH as the source of electrons. The oxidation of lauric acid by the reconstituted system increased 16-fold as the temperature increased from 25 to 70 degrees C and was functional for more than 30 min at the higher temperature. This first in vitro high-temperature P450 catalytic system is a key step in the development of practical high-temperature monooxygenase systems.

Mitochondrial cytochrome P450 27A1 (P450 27A1) catalyzes 27-hydroxylation of cholesterol, the first step in the alternative bile acid biosynthetic pathway. Although several crystal structures of P450s are known, no structural information is available for the mammalian, membrane-bound enzymes involved in the removal of cholesterol from the body. We prepared a three-dimensional model of P450 27A1 based on the structure of P450 BM-3. Conservative and non-conservative mutations were introduced at hydrophobic and positively charged residues in the putative F-G loop and the adjacent helix G (positions 219-237). Subcellular distribution of the mutant P450s expressed in Escherichia coli was used as a measure of membrane-protein interactions. Conservative substitutions of residues located on the surface, according to our model, L219V, L219I, Y220F, F223Y, L224I, R229K, V231L, F234Y, K236R, and R237K, weakened the association of the mutant P450s with the membrane and led to the appearance of up to 21% of P450 27A1 in the bacterial cytosol. It is likely that the mutated side chains are involved in binding to membrane phospholipids. Substitutions in the F-G loop did not significantly affect the K(m) value for cholesterol hydroxylation. However, non-conservative mutants, L219N, Y220A, Y220S, F223A, K226R, and R229A, had significantly impaired catalytic properties, indicating strict requirements for the size and polarity of the side chains at these positions for the catalysis. The results provide insight into the membrane topology of mitochondrial P450s and indicate the importance of membrane-protein interactions in the efficiency of reactions catalyzed by P450 27A1.

The kinetic characteristics (kcat, Km, and their ratio) for oxidation of iodide (I-) at 25 degrees C in 0.2 M acetate buffer, pH 5.2, and tetramethylbenzidine (TMB) at 20 degrees C in 0.05 M phosphate buffer, pH 6.0, with 10% DMF catalyzed by human thyroid peroxidase (HTP) and horseradish peroxidase (HRP) were determined. The catalytic activity of HRP in I- oxidation was about 20-fold higher than that of HTP. The kcat/Km ratio reflecting HTP efficiency was 35-fold higher in TMB oxidation than that in I- oxidation. Propyl gallate (PG) effectively inhibited all four peroxidase processes and its effects were characterized in terms of inhibition constants Ki and the inhibitor stoichiometric coefficient f. For both peroxidases, inhibition of I- oxidation by PG was characterized by mixed-type inhibition; Ki for HTP was 0.93 microM at 25 degrees C. However, in the case of TMB oxidation the mixed-type inhibition by PG was observed only with HTP (Ki = 3.9 microM at 20 degrees C), whereas for HRP it acted as a competitive inhibitor (Ki = 42 microM at 20 degrees C). A general scheme of inhibition of iodide peroxidation containing both enzymatic and non-enzymatic stages is proposed and discussed.

The substituted pyrocatechols sodium (2,3-dihydroxy-4,6-di-tert-butyl-phenyl)-S-thiosulfate (InH1) and 3-(S-glutathionyl)-4,6-di-tert-butyl-pyrocatechol (InH2) effectively inhibited oxidation of o-phenyle-nediamine and tetramethylbenzidine (TMB) catalyzed by hemin or its complex with BSA (methemalbumin). The method of competitive reactions of aromatic amines (PDA, TMB) and pyrocatechols (InH1, InH2) with active radicals was used for the quantitative determination of rates of radical initiation in systems containing hemin (methemalbumin) and H2O2 (TBHP). The use of the inhibitor method for heme-protein-peroxide systems is complicated by heterogeneity of the radicals generated (HO., RO., HO2., RO2., peroxide-like compounds I and II), formation of complexes of the inhibitors with protein component, and possible interaction of active radicals with organic co-solvents (DMF or DMSO). The system hemin (methemalbumin)-H2O2 (TBHP)-aromatic amines can be used for testing the efficacy of potential bioantioxidants.