Novel reactivity of cytochrome P-450-CAM. Methyl hydroxylation of 5,5-difluorocamphor.

The interaction of the camphor hydroxylating P-450 isolated from Pseudomonas putida grown on camphor (P-450-CAM) with 5,5-difluorocamphor, a substrate analog in which the two methylene hydrogens at the normal site of hydroxylation have been replaced with fluorine, has been examined. This compound binds tightly to the enzyme with a dissociation constant and UV-visible absorption spectrum identical to that observed with d-camphor. In the presence of the reconstituted P-450-CAM system, 5,5-difluorocamphor is metabolized at a rate approximately one-third the rate of the physiological substrate, d-camphor, resulting in the formation of a hydroxylated product with a molecular weight of 204 as well as a minor (less than 3%) hydroxylated product of molecular weight 184. Isotopically labeled molecular oxygen (18O2) is incorporated into the major product while labeled oxygen from water (H218O) is not incorporated, clearly indicating that the hydroxyl oxygen originates from dioxygen. Proton NMR characterization (400 MHz) of the major product has led to its assignment as 5,5-difluoro-9-hydroxy-camphor, with supporting structural evidence provided by the mass spectral fragmentation pattern. The formation of 9-hydroxylated product represents the first example of methyl hydroxylation catalyzed by cytochrome P-450-CAM, indicates a change in regio-selectivity when the normal site of reaction is blocked, and supports the hypothesis that the delivery of the oxygen atom occurs from the exo side of the camphor molecule.

NADH- and oxygen-dependent multiple turnovers of cytochrome P-450-CAM without putidaredoxin and putidaredoxin reductase.

Phenazine methosulfate (PMS) has been successfully used to mediate electron transfer from NADH to cytochrome P-450-CAM in the absence of putidaredoxin and putidaredoxin reductase under aerobic conditions. Identification and quantitation of exo-5- hydroxycamphor , the only product, has been accomplished by gas chromatography. In the absence of cytochrome P-450-CAM, or when other heme proteins (hemoglobin, myoglobin, horseradish peroxidase) are substituted for P-450-CAM, no exo-5- hydroxycamphor is detected. Product formation is not inhibited by the addition of catalase, superoxide dismutase, or hydroxyl radical scavengers; however, significant inhibition is observed with carbon monoxide and metyrapone, known inhibitors of the fully reconstituted P-450 system. Addition of 2,3-dimercaptopropanol to the NADH/PMS/P-450 system leads to a 4-fold increase in product formation; when putidaredoxin is added (without dimercaptopropanol), a 20-fold increase in product formation is observed. Constant bubbling with oxygen results in a further increase in the amount of product (150-fold increase overall). Our results show that PMS can substitute for the electron-transfer proteins putidaredoxin and putidaredoxin reductase in the transfer of electrons from NADH to P-450-CAM, resulting in multiple turnovers. Molecular oxygen dependent multiple turnovers of cytochrome P-450 have not been previously observed without the fully reconstituted, three-protein system.

Reconstitution of mitochondrial F0.F1-ATPase with phosphatidylcholine using the nonionic detergent, octylglucoside.

A reconstitution procedure has been developed for the incorporation of the mitochondrial F0.F1-ATPase into the bilayer of egg phosphatidylcholine vesicles. The nonionic detergent, octylglucoside, egg phosphatidylcholine, and the lipid-deficient, oligomycin-sensitive F0.F1-ATPase (Serrano, R., Kanner, B., and Racker, E. (1976) J. Biol. Chem. 251, 2453-2461) were combined in a 4770:320:1 detergent/phospholipid/protein molar ratio and then centrifuged on a discontinuous sucrose gradient to isolate the F0.F1-phosphatidylcholine complex. The specific activity of the reconstituted F0.F1-ATPase was as high as 14.5 mumol/min/mg protein, whereas with no added lipid the activity ranged between 1.4 and 2.2 mumol/min/mg protein. This reconstituted preparation exhibited greater than 90% oligomycin sensitivity which demonstrated the intactness of the multisubunit enzyme complex. The phosphatidylcholine/protein molar ratio of the reconstituted F0.F1 was 250:1 with less than 0.4% of the added octylglucoside remaining. Titrations with both phosphatidylcholine and octylglucoside demonstrated that the specific activity and oligomycin sensitivity were highly dependent on the concentrations of both phospholipid and detergent in the original reconstitution mixture. Analysis of the reconstituted ATPase by electron microscopy demonstrated that the catalytic portion of the enzyme complex projected from the phospholipid bilayer with an orientation similar to that observed with submitochondrial particles. The F0.F1-phosphatidylcholine complex was able to trap inulin, which suggests a vesicular structure impermeable to macromolecules. The electrophoretic mobility of the complex was identical to that for liposomes of egg phosphatidylcholine alone. The reconstitution conditions utilized give rise to an enzyme-phospholipid complex with very low ionic charge that demonstrates high oligomycin-sensitive ATPase activity.

Preparation and properties of ferrous chloroperoxidase complexes with dioxygen, nitric oxide, and an alkyl isocyanide. Spectroscopic dissimilarities between the oxygenated forms of chloroperoxidase and cytochrome P-450.

Extensive spectroscopic investigations of chloroperoxidase and cytochrome P-450 have consistently revealed close similarities between these two functionally distinct enzymes. Although the CO-bound ferrous states were the first to display such resemblance, additional comparisons have focused on the native ferric and ferrous and the ligand-bound ferric derivatives of the enzymes. In order to test the extent to which the spectral properties of the two enzymes match each other, we have prepared the NO, alkyl isocyanide, and O2 adducts of ferrous chloroperoxidase, the latter two for the first time. As expected, the NO adducts of the two proteins have similar UV-visible absorption and magnetic circular dichroism spectra; the same behavior is observed for the alkyl isocyanide complexes. Unexpectedly, the dioxygen adduct of ferrous chloroperoxidase (i.e. Compound III), generated in cryogenic solvents at -30 degrees C by bubbling with O2, is spectrally distinct from oxy-P-450-CAM. Identification of this derivative as oxygenated chloroperoxidase is based on the following criteria: It is EPR-silent at 77 K. The bound O2 is dissociable as judged by the uniform conversion to the CO-bound form. Oxy-chloroperoxidase autoxidizes to form the native ferric enzyme without detectable intermediates at a rate comparable to that determined for oxy-P-450-CAM. Oxy-chloroperoxidase exhibits optical absorption (lambda nm (epsilon mM) = 354 (41), 430 (94), 554 (16.5), 587 (12.5)) and magnetic circular dichroism spectra that are clearly distinct from those of histidine-ligated heme proteins such as oxy-myoglobin or oxy-horseradish peroxidase. Surprisingly, several of its spectral properties, namely the red-shifted Soret peak and discrete alpha peak, are also unlike those of oxy-P-450-CAM. Since considerable evidence has accumulated supporting the ligation of an endogenous thiolate to the heme iron of chloroperoxidase, as has been established for the P-450 enzyme, the observed dissimilarities suggest that the electronic properties of the two dioxygen adducts are quite sensitive to differences in their active site heme environment. This, in turn may be related to the functional differences between the two enzymes.

Tightly associated cardiolipin in the bovine heart mitochondrial ATP synthase as analyzed by 31P nuclear magnetic resonance spectroscopy.

The bovine heart F0F1-ATPase preparation (Serrano, R., Kanner, B., and Racker, E. (1976) J. Biol. Chem. 251, 2453-2461) has been further delipidated. The lipid-deficient preparation contained 2.5 mol of cardiolipin, 1 mol of phosphatidylcholine (PC), and 1 mol of phosphatidylethanolamine (PE) per mol of F0F1. When reconstituted with asolectin the delipidated preparation exhibited an activity of 13 mumol of ATP hydrolyzed/min/mg of protein which was 88% oligomycin-sensitive. The phospholipids in this preparation were analyzed by 31P NMR spectroscopy to determine if they were immobilized by the enzyme (rendered NMR-invisible). The PC and PE were below the limits of detection under the conditions utilized and the cardiolipin was NMR-invisible until the enzyme was denatured by addition of either 1% sodium dodecyl sulfate or 8 M urea. Addition of cardiolipin to the delipidated preparation and subsequent analysis by NMR spectroscopy revealed that approximately 4 mol of cardiolipin were immobilized per mol of F0F1 ATPase. The enzyme appears to have high affinity for cardiolipin exclusively, since PC (a prominent inner membrane lipid), phosphatidyl serine (an acidic phospholipid), and phosphatidyl glycerol (the precursor to cardiolipin) were not immobilized (rendered NMR-invisible) when added to the delipidated preparation.

Ethanol-elicited alterations in the oligomycin sensitivity and structural stability of the mitochondrial F0 . F1 ATPase.

Liver mitochondria from rats fed ethanol chronically demonstrated a 35% decrease in mitochondrial ATPase activity. Moreover, the ATPase activity was inhibited only 61% by addition of oligomycin. Treatment of mitochondria from ethanol-fed rats with the detergent, Lubrol-WX, caused the release of 36% of the F1 from the resulting inner membrane particles. In comparison, only 5% of the F1 was dissociated when control mitochondria were subjected to the Lubrol treatment. However, when the units of ATPase activity from the supernatant and particles obtained after Lubrol treatment were added together, their sums were equivalent in preparations from control and ethanol-fed animals. Moreover, polyacrylamide gel electrophoresis analyses indicated equal amounts of the alpha + beta subunits of F1 in mitochondria from control and ethanol-fed rats. Reconstitution experiments with urea particles and F1 prepared from both control and ethanol mitochondria revealed a decrease in oligomycin sensitivity which could be attributed to an alteration in the functioning of either the oligomycin sensitivity conferring protein or a membrane sector subunit that interacts with oligomycin. Analysis by reconstitution also demonstrated that there were no ethanol-elicited alterations in the properties of the F1 portion of the ATP synthase complex. These observations indicate that the activity of the ATP synthase complex is altered significantly by ethanol-elicited changes in the functioning of those polypeptides involved in modulating both oligomycin sensitivity and the association of F1 with membrane sector subunits.

X-ray absorption spectroscopic studies of high valent iron porphyrins. Horseradish peroxidase compounds I and II and synthetic models.

X-ray absorption edge and extended x-ray absorption fine structure studies have been undertaken on resting (ferric) horseradish peroxidase, HRP compound I (HRP-I), HRP compound II (HRP-II), and several highly oxidized synthetic iron porphyrins that may have relevance as models for the iron site in horseradish peroxidase. The energies of the absorption edges are consistent with an Fe(IV) formulation for the highly oxidized species. The shapes of the absorption edges further support the assignment of HRP-I and one of the model compounds as Fe(IV)-porphyrin pi-cations. The edge shapes also demonstrate that the iron sites in the model porphyrins are not identical to the iron sites in the enzyme. Preliminary curve fitting analysis of the extended x-ray absorption fine structure clearly indicates the presence of two different nearest neighbor distances for the iron, both in HRP-I and HRP-II, as well as in two of the highly oxidized model porphyrins. These distances are interpreted as an iron-porphyrin nitrogen distance and as a short (approximately 1.6 A) iron-oxygen distance.