©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Uncoupling Oxygen Transfer and Electron Transfer in the Oxygenation of Camphor Analogues by Cytochrome P450-CAM
DIRECT OBSERVATION OF AN INTERMOLECULAR ISOTOPE EFFECT FOR SUBSTRATE C-H ACTIVATION (*)

(Received for publication, August 14, 1995)

Saloumeh Kadkhodayan (1) Eric D. Coulter (1) David M. Maryniak (1) Thomas A. Bryson (1) John H. Dawson (1) (2)(§)

From the  (1)Department of Chemistry and Biochemistry and the (2)School of Medicine, University of South Carolina, Columbia, South Carolina 29208

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The hydroxylation of (1R)-camphor by cytochrome P450-CAM involves almost complete coupling of electron to oxygen transfer. Modifications at C-5 of camphor, the normal site of hydroxylation by P450-CAM, lead to as much as 98% uncoupling of electron and oxygen transfer as well as to decreases in the rate of electron uptake (up to 10-fold) and the rate of oxygenated product formation (up to 210-fold). Two modes of uncoupling are seen: (a) two-electron uncoupling in which the decrease in oxygenated product formation is balanced by increases in H(2)O(2) formation and (b) four-electron ``oxidase'' uncoupling where the NADH/O(2) ratio has changed from one to nearly two and relatively little H(2)O(2) is formed. Both enantiomers of 5-methylenylcamphor are two-electron uncouplers, while (1R)- and (1S)-5,5-difluorocamphor and (1R)-9,9,9-d(3)-5,5-difluorocamphor are four-electron uncouplers. An intermolecular isotope effect of 11.7 is observed for oxygenation of C-9 in (1R)-5,5-difluorocamphor. With this substrate, the significant decrease in the rate of oxygenated product formation combined with the large isotope effect suggest that the rate-limiting step has switched from electron to oxygen transfer.


INTRODUCTION

The cytochromes P450 (P450) (^1)are a family of b-type heme monooxygenases that catalyze the activation of dioxygen for incorporation into a variety of organic substrates(1, 2, 3, 4, 5, 6, 7) . Much of our present knowledge of the structure and mechanism of P450 enzymes comes from investigations of the bacterial P450-CAM isolated from Pseudomonas putida. P450-CAM catalyzes the highly stereoselective hydroxylation of its physiological substrate, (1R)-camphor, to form (1R)-5-exo-hydroxycamphor as the sole product (Fig. R1). In addition, the enzyme will hydroxylate related bi- and tricyclic molecules such as norcamphor(8, 9, 10) , thiocamphor(11) , adamantane(12) , adamantanone(12) , camphane(11) , and 5,5-difluorocamphor (Fig. R1) (13) as well as epoxidize 5,6-dehydrocamphor (14) and 5-methylenylcamphor (Fig. R1)(15) . Both enantiomers of camphor and 5-methylenylcamphor are oxygenated by the enzyme(15) . Recently, it has been found to be capable of hydroxylating and epoxidizing a variety of molecules that bear no structural resemblance to camphor such as ethylbenzene(16) , nicotine(17) , 4-methyl-1-tetralone(18) , styrene(19) , 1,1,1-trichloroethane(20) , thioanisole(21) , and several others(22) .


Figure R1: Reactions 1-3.



Four well characterized intermediates (1-4, Fig. 1) have been observed in the reaction cycle of P450(1, 2, 3, 4, 5, 6, 7) . The low-spin ferric resting state (1) becomes high-spin (2) upon substrate binding. Electron transfer from NADH via two-electron transfer proteins, putidaredoxin reductase (PdR) and putidaredoxin (Pd), yields the high-spin deoxyferrous state (3) that is capable of binding O(2) to generate the oxyferrous state (4) or CO to produce the carbonmonoxy ferrous inhibitor adduct (5). Addition of the second electron, the rate-limiting step in the cycle(23) , is proposed to yield a ferric peroxide adduct (6a) which can be protonated on oxygen to give a hydroperoxide complex (6b). Protonation of the oxygen that already bears a proton and heterolytic cleavage of the peroxide O-O bond releases water and generates the proposed oxo-ferryl porphyrin radical intermediate 7. Intermediate 7 then transfers an oxygen atom to the hydrocarbon substrate, presumably by an oxygen rebound mechanism(24) , to give the alcohol product and the P450 state 1. With (1R)-camphor, the cycle just described proceeds with nearly complete coupling (9, 15) of electron transfer (2 3 and 4 6) to oxygen transfer (7 1). Two modes of uncoupling electron transfer from oxygen transfer after addition of the second electron have been described(8, 16, 25) . Protonation of 6b on the unprotonated oxygen would lead to release of hydrogen peroxide generating 2 and no oxygenated product (Path A, 6b 2, Fig. 1, two-electron uncoupling). Alternatively, reduction of 7 by two electrons and concurrent protonation would give a second molecule of water (the ``oxidase'' reaction), regenerate 2 and produce no oxygenated product (Path B, 7 2, Fig. 1, four-electron oxidase uncoupling).


Figure 1: Reaction cycle of cytochrome P450. RH represents the substrate (camphor or camphor analog) and R(O)H represents the oxygenated (hydroxylated or epoxidized) product. The dianionic porphyrin macrocycle is abbreviated as a parallelogram with nitrogens at the corners. Oxy-P450 (4) is shown as a complex of ferric porphyrin and superoxide anion, but could also be described as an adduct of neutral dioxygen and ferrous porphyrin. States 6a, 6b, and 7 are hypothetical intermediates whose structures have not been established. Structures 1, 2, and 7 are neutral (the dot and the positive charge on 7 indicate the radical state and electron deficiency of the -electron system of the porphyrin ring), while the overall charge on structures 3, 4, 5, and 6b is -1 and on intermediate 6a is -2. Two modes of uncoupling electron and oxygen transfer, A and B, are shown. See text for further discussion of the events in the cycle and the nature of the numbered intermediates.



In an effort to probe the mechanism of dioxygen activation by P450-CAM through blockage of the normal site of reaction, we have synthesized camphor analogues with two fluorine atoms or an exocyclic olefin functionality at C-5. We have previously reported that 5,5-difluorocamphor is hydroxylated at C-9 to give the hydroxymethyl product (13) and that 5-methylenylcamphor is epoxidized to give the exo-epoxide product(15) . Herein we describe in detail the substrate binding constants along with the electron, dioxygen, and substrate stoichiometries for the reactions of both (1R)- and (1S)-enantiomers of the difluoro and methylenyl substrates as well as for (1R)-9,9,9-d(3)-5,5-difluorocamphor. Parallel studies have been carried out with both (1R)- and (1S)-camphor. The difluoro and methylenyl substrates are oxygenated at appreciably slower rates than (1R)-camphor. This may reflect a change in the rate-limiting step from the second electron transfer to a later step, possibly oxygen transfer. In addition, oxygen transfer to the camphor analogues is uncoupled from electron transfer to such an extent that in many cases, the majority of the electrons that flow into the system do not participate in the production of oxygenated product. Finally, the two types of substrates are uncoupled in different ways: the difluoro substrates are four-electron uncouplers while the methylenyl substrates are two-electron uncouplers.


MATERIALS AND METHODS

Protein Purification

P450-CAM, Pd, and PdR were isolated from P. putida grown on (1R)-camphor. P450-CAM was purified by the method of Peterson and co-workers (26) with minor modifications(27) . Pd and PdR were purified by published procedures (28) .

Synthesis of Camphor Analogues

(1R)- and (1S)-camphor were purchased from Aldrich and used without further purification. The syntheses of (1R)- and (1S)-methylenylcamphor(15) , (1R)-5,5-difluorocamphor(13) , (1S)-5,5-difluorocamphor, and (1R)-9,9,9-d(3)-5,5-difluorocamphor (29) have been described elsewhere.

Dissociation Constant Determination

Dissociation constants (K(d)) were determined at 4 °C as described previously (13) by titration of substrate-free P450-CAM (1.5 µM) with an aqueous 3-5 mM solution of substrate in 20 mM phosphate buffer containing KCl (100 mM). Binding was followed by monitoring the decrease in absorbance at 417 nm.

Enzyme Activity

Catalytic activities were determined from the rate of NADH oxidation. This was monitored as a loss of absorbance at 340 nm ( = 6.22 mM cm) as a function of time. Typical assay mixtures contained P450-CAM (0.05 µM), PdR (4.0 µM), Pd (10.0 µM), substrate (1.0 mM), NADH (0.5 mM), and KCl (100 mM) in 20 mM phosphate buffer at 25 °C. Protein concentrations were chosen to optimize turnover rate.

Product Formation and Uncoupling

Reaction mixtures contained P450-CAM (0.5 µM), PdR (4.0 µM), Pd (10 µM), substrate (1.0 mM), and NADH (1.0 mM) with oxygen as the limiting reagent. NADH consumption was monitored as described above. Reactions were allowed to proceed for 15 min at which time a known amount of internal standard, 3-endo-bromocamphor, was added and the products extracted twice with CH(2)Cl(2). The organic extract was concentrated under a slow stream of N(2) and analyzed by gas chromatography and gas chromatography/mass spectrometry. Uncoupling (H(2)O(2) and/or H(2)O formed) was calculated based on the difference between NADH consumed and product formed.

Oxygen Consumption

Oxygen consumption was continuously measured using a Clark-type electrode in conjunction with a Gilson 5/6 oxygraph. Concentrations for P450-CAM, Pd, PdR, NADH, and substrate are as specified under product formation and uncoupling. A full-scale deflection of approximately 280 µmol/ml was determined by the method of Robinson and Cooper(30) . NADH oxidation was simultaneously monitored as above.

Determination of Hydrogen Peroxide Formation

Reaction concentrations were identical for all components to those described under the product formation and uncoupling section in a total volume of 3.0 ml. H(2)O(2) production was monitored at time intervals by removing portions of the reaction mixture, quenching with 3% aqueous trichloroacetic acid, and performing a colorimetric determination at 480 nm as described by Atkins and Sligar(10) . Values of H(2)O(2) produced were derived from comparison to a standard curve prepared under identical conditions and are for 15-min reaction periods.

Instrumentation

Varian-Cary 210 or 219 spectrophotometers were used for UV-visible absorption spectral analysis. A Hewlett-Packard 5890A gas chromatograph with a stabilwax column (30 meter, 0.25-mm inner diameter) programmed at 70 °C followed by a temperature increase to 225 °C at 10 °C/min and equipped with a Hewlett-Packard 3390A reporting integrator was used for product analysis. Electron impact mass spectra and gas chromatography/mass spectral data were obtained on a Finnegan Model 4521 spectrometer. High resolution mass spectrometry was performed on a VG Instruments 70SQ spectrometer.


RESULTS AND DISCUSSION

Binding Characteristics of Camphor Analogues

The major structural feature which distinguishes camphor-free cytochrome P450-CAM from the camphor-bound enzyme is the presence of six water molecules in the substrate binding pocket in the absence of camphor(31) . Substrate binding expels these water molecules (31, 32) and converts the ferric heme iron from low-spin six-coordinate water-ligated (1, Fig. 1) to high-spin five-coordinate (2)(31, 32) . The low- to high-spin conversion results in a shift of the Soret peak in the UV-visible absorption spectrum from 417 to 391 nm and the appearance of a peak at 646 nm. The absorption spectra of ferric P450-CAM with (1R)-camphor and (1S)-camphor bound are displayed in Fig. 2. The symmetrical Soret peak observed in the presence of (1R)-camphor is consistent with complete low- to high-spin conversion, whereas incomplete spin state conversion is indicated for the (1S)-camphor-bound enzyme by the shoulder seen near 417 nm and the diminished intensity at 646 nm. Incomplete conversion of the heme iron from low- to high-spin upon substrate binding, as seen with norcamphor and 5-bromocamphor, can be attributed to the inability of these substrates to completely displace the distal water ligand(33, 34) . Identical spectra to those displayed in Fig. 2for the (1R)- and (1S)-camphor-bound enzyme were observed with all the other (1R)- and (1S)-substrates, respectively (data not shown). These results indicate that the heme remains partially hexa-coordinate in the presence of the (1S)-substrates, but becomes penta-coordinate upon addition of the (1R)-substrates. Differences are also seen in the binding constants for the (1R)- and (1S)-substrates (Table 1). The K(d) values for the (1R)-substrates including (1R)-camphor itself are between 1.2 and 1.8 µM, whereas the K(d) values of the (1S)-enantiomers are 1.5-7-fold higher (weaker binding). As judged by the absorption spectra of the substrate-bound enzyme and the binding dissociation constants, the (1R)-camphor analogues bind to ferric P450-CAM in an identical manner to (1R)-camphor, whereas the (1S)-camphor analogues bind more weakly and do not completely exclude water from remaining bound to a fraction of the active site heme iron centers. Clearly, the (1S)-camphor analogues do not bind optimally to the enzyme.


Figure 2: Electronic absorption spectrum of (1R)-camphor bound (solid) and (1S)-camphor bound (dashed) cytochrome P450-CAM. Protein concentrations were 70 and 40 µM, respectively. Spectra were recorded at 4 °C in 20 mM phosphate buffer, pH 7.4, containing 100 mM KCl and under conditions of saturating substrate concentration (1 mM).





Analysis of Oxygenated Products

The oxygenation of the (1R)- and (1S)-enantiomers of camphor, 5,5-difluorocamphor, and 5-methylenylcamphor, and of (1R)-9,9,9-d(3)-5,5-difluorocamphor by the fully reconstituted NADH/PdR/Pd/P450-CAM system has been analyzed by gas chromatography, mass spectrometry, and NMR spectroscopy. (1R)-Camphor forms exclusively the 5-exo-alcohol (Fig. R1), (1R)-5,5-difluorocamphor yields primarily the 9-alcohol (Fig. R1), and (1R)-5-methylenylcamphor produces the exo-epoxide (Fig. R1)(15) . Table 2contains a summary of the oxygenated products formed and their percent distribution. The absolute amount of hydroxylated camphor product formed with each substrate is dependent on the degree of coupling (Table 1) and will be discussed below; with some substrates, the uncoupling products (hydrogen peroxide and, especially, water) are the major reaction products. (1S)-Camphor and (1S)-5-methylenylcamphor produce predominantly the same organic product as their (1R)-enantiomers. With (1S)-5,5-difluorocamphor, however, the primary hydroxylated product is not the same as is formed with the (1R)-enantiomer. The new product is assigned to be 3-hydroxy-5,5-difluorocamphor based on the prominant (M - 28) ion in the mass spectrum that is typical of 3-hydroxybornanones(11) . However, because of the small amount of this product obtained, its proposed structure has not been confirmed by NMR analysis. All three fluorinated substrates produce an additional product that we propose to be 6-hydroxy-5,5-difluorocamphor based largely on the lack of (M - 28) and (M - 31) peaks characteristic of 3-hydroxy substituted camphors (11) and hydroxymethyl-substituted camphors(13) , respectively. Once again, insufficient quantities were obtained for NMR analysis. The proposed 6-alcohol product is only a minor component with both enantiomers of 5,5-difluorocamphor, but it is formed in an equal amount to the 9-alcohol in the case of (1R)-9,9,9-d(3)-5,5-difluorocamphor. This shift in the ratio of oxygenation products from 92:8 (9-alcohol:6-alcohol) in the d(0) case to 50:50 (as well as increased water production) in the d(3) case presumably results from metabolic switching due to the deuterium isotope effect. Similar observations have been made by Atkins and Sligar (8) with deuterated norcamphor.



In analyzing the crystal structures of both camphor-bound and camphor-free P450-CAM, Poulos and co-workers (31, 32, 33) have suggested that substrate specificity is achieved by specific interactions between the substrate, (1R)-camphor, and appropriately positioned amino acids in the substrate binding pocket. The most important interactions are a hydrogen bond between Tyr and the carbonyl oxygen on C-2 of camphor and van der Waals contacts between Val and the 8,9-geminal dimethyl groups of camphor and between Leu-Val and the 10-methyl group of camphor. Like (1R)-camphor, (1S)-camphor reacts exclusively at the 5-exo-position. This suggests that the binding position of (1S)-camphor may still be oriented by the hydrogen bond between its carbonyl group and Tyr. To maintain this hydrogen bond and also position C-5 in the same relative position as in (1R)-camphor requires that the protons on C-3 in (1S)-camphor occupy the position where the 10-methyl group was located in (1R)-camphor (Fig. S1). This modifies the protein-substrate van der Waals interactions; the incomplete spin shift upon binding (1S)-camphor indicates that the binding interaction is not optimal. Nonetheless, hydroxylation of (1S)-camphor still occurs at the exo position of C-5. The same arguments regarding regio- and stereochemistry would seem to apply to the epoxidation of (1R)- and (1S)-5-methylenylcamphor. Interestingly, a recent study of the hydroxylation of (1R)- and (1S)-norcamphor showed that while C-5 is the primary site of hydroxylation for the (1R)-enantiomer, C-6 was preferred for (1S)-norcamphor(9) .


Figure S1: Scheme 1.



For 5,5-difluorocamphor, blocking C-5 with fluorines shuts off reaction at that carbon completely. The major product in the 1R case is the 9-alcohol. This is not surprising since C-9 is the second closest carbon to the heme iron after C-5(32, 36) . For the (1S)-enantiomer, the non-optimal binding and slow rate of oxygen transfer (Table 3) lead to multiple reaction sites including the only example of C-3 hydroxylation observed herein (Table 2).



NADH Oxidation Rates

As both electrons in the P450 cycle (Fig. 1) are ultimately derived from NADH, the rate of NADH oxidation reflects the rate of enzyme turnover when electron transfer is tightly coupled to oxygen transfer as with (1R)-camphor. However, as discussed above, uncoupling can occur at internal branch points in the reaction cycle; two-electron uncoupling (Path A, Fig. 1) involves release of hydrogen peroxide while four-electron uncoupling (Path B, Fig. 1) leads to production of a second molecule of water(8, 16, 25) . Therefore, the rate of NADH oxidation seen with such substrates not only reflects the diminished rate of oxygenated product formation but also the amount of water and hydrogen peroxide generated through uncoupling. The kinetic and stoichiometric data for the reaction of (1R)- and (1S)-camphor and for the five camphor analogues with the fully reconstituted NADH/PdR/Pd/P450-CAM system are summarized in Table 1. The NADH oxidation rate for (1S)-camphor is approximately half the rate of the (1R)-enantiomer. However, in the cases of the methylenyl and difluoro substrates where the five position has either been modified or blocked, the rate of (1S)-enantiomers are about twice those of the (1R)-enantiomers. Looked at in a different way, fluorination of the substrate causes the NADH oxidation rate to decrease much more significantly within the 1R series (camphor, 5,5-difluorocamphor, and 5-methylenylcamphor) than within the 1S series.

With (1R)-9,9,9-d(3)-5,5-difluorocamphor, the rate of NADH oxidation (557 min) is actually higher than the rate for the non-deuterated substrate (420 min) (Table 1). Similar apparent inverse isotope effects have been reported for norcamphor by Sligar and co-workers(8) . Once again, however, it is important to remember that with uncoupled substrates, the NADH oxidation rate reflects not only diminished oxygenated product formation but also two- and four-electron uncoupling. From the uncoupling and stoichiometry data in Table 1, it is possible to calculate the rates of formation of oxygenated products (Table 3). This analysis shows that the 9,9,9-d(3)-substrate produces oxygenated products at a much slower rate than undeuterated 5,5-difluorocamphor. The apparent inverse isotope effect for NADH oxidation rates for the 9,9,9-d(3)-substrate clearly results from the significantly increased amount of four-electron uncoupling observed with that substrate. The data in Table 3also reveal that the rate of oxygenated product formation can be decreased by as much as 210-fold upon modification of the substrate.

To factor out the effects of four-electron uncoupling, it is convenient to define an O(2) turnover number by dividing the NADH oxidation rate by the NADH/O(2) consumption ratio (Table 1). This provides a turnover number based on O(2) consumption. With one O(2) consumed per cycle of the enzyme, this value represents the number of cycles the enzyme carries out per minute. From these data, it is seen that d(0)- and d(3)-(1R)-difluorocamphor each turn over about 10-fold more slowly than (1R)-camphor.

Intermolecular Deuterium Isotope Effects

The rates in Table 3are for the sum of the various products produced from a given substrate. These rates are determined under optimized turnover conditions which require a very large excess of Pd and PdR. Comparison of the rates of oxygenated product formation for 5,5-difluorocamphor versus the 9,9,9-d(3) molecule reveals an isotope effect of 6.4 (Table 4, part A). However, as mentioned above, the presence of the deuteriums on C-9 leads to a higher percentage of oxygenation on C-6. Recalculation of the isotope effect for d(0)- and d(3)-5,5-difluorocamphor based solely on hydroxylation at C-9 (Table 4, part A) leads to an isotope effect of 11.8. The isotope effect can be determined in a second way by using the quantities of products formed (Table 1) upon oxygenation of d(0)- and d(3)-5,5-difluorocamphor under conditions where oxygen is the limiting reagent (and without as large an excess of Pd and PdR). As above, the amounts of products formed specifically at C-6 and C-9 can be calculated using the product distribution data in Table 2. After that adjustment, the ratio of the amounts of oxygenated products formed at C-9 for the d(0) and d(3) substrate is 11.7 (Table 4, part B). In both experiments, the reactivity at C-6 is unaffected by the presence or absence of deuteriums at C-9 (Table 4, parts A and B).



With 5,5-difluorocamphor, the significant slowdown in the rate of formation of oxygenated products (Table 3) suggests that the rate-limiting step in the P450 cycle may have changed from electron transfer to oxygen transfer (which in turn is initiated by hydrogen atom abstraction). This conclusion is strengthened by the intermolecular isotope effect of 11.7 for hydroxylation of C-9 in 5,5-difluorocamphor (Table 4). With 5-exo- and 5-endo-deuterated camphors, Gelb et al.(37) reported an intermolecular isotope effect of only 1.1-1.2 and concluded that oxygen transfer is sufficiently fast that the intrinsic isotope effect, k(H)/k(D), for hydrogen atom removal is masked. The intermolecular isotope effect of 11.7 for oxygenation of C-9 in 5,5-difluorocamphor matches the intramolecular values of 11 or greater reported by Hjelmeland et al.(38) and Groves et al.(39) for aliphatic hydroxylation by liver microsomal P450. The fact that the intermolecular isotope effect is no longer masked suggests that the rate of hydrogen atom abstraction, which initiates oxygen atom transfer, has been slowed down sufficiently at C-9 in 5,5-difluorocamphor to replace the second electron transfer as the rate-limiting step for oxygenation at that site.

Reaction Stoichiometries and Uncoupling Reactions

Important additional information about the nature of the uncoupling of electron transfer and oxygen transfer is revealed by examination of the reactions between the substrates and the NADH/PdR/Pd/P450-CAM system under conditions where the limiting reagent is dioxygen. (1R)-Camphor clearly exhibits a 1:1:1 ratio of NADH oxidized to O(2) consumed to product formed (Table 1). This is no longer true for (1R)-5-methylenylcamphor. Although the ratio of NADH oxidized to O(2) consumed essentially remains 1:1 (1.07, Table 1), the relative amount of oxygenated product formed is now less than 1. The system is uncoupled with increased levels of hydrogen peroxide making up for the decrease in oxygenated product formed. Interestingly, (1S)-5-methylenylcamphor has a rate of NADH consumption that is over 5-fold slower than (1R)-camphor, but it is only 9% uncoupled.

With the three difluoro substrates, the average amount of NADH oxidized is about 440 nmol/ml while the consumption of dioxygen remains about 260 nmol/ml. The amount of oxygenated product formed was also considerably lower, the system now being 83-98% uncoupled. The NADH/O(2) consumption ratio (Table 1) approaches 2.0 with increasing uncoupling. Relatively little hydrogen peroxide is formed. These observations combined with the apparent inverse deuterium isotope effect discussed earlier are consistent with four-electron oxidase uncoupling for the difluorocamphor substrates.

The exact causes for the uncoupling of the P450 reaction cycle are not known. Extensive studies by Raag and Poulos (33, 40) have failed to attribute it to any one factor. Uncoupling may result from less complementary enzyme-substrate fit or from the lack of the hydrogen bond between the substrate carbonyl and Tyr since the hydroxylation of norcamphor and camphane, respectively, by P450-CAM are substantially uncoupled(11, 41) . Uncoupling is not related to the fraction of low-spin ferric heme, since thiocamphor-bound P450-CAM and the camphor-bound Tyr-Phe P450-CAM mutant are only 65 and 59% high-spin, respectively, yet have a very high hydroxylation efficiency (100 and 98%, respectively)(11) . An important factor that promotes uncoupling seems to be the presence of extra solvent water near the O-O bond undergoing cleavage. To avoid the two modes of uncoupling requires the precise delivery of two protons exclusively to the outer oxygen of the iron-bound peroxide to generate the oxo-ferryl state (6a 6b 7) and the prevention of electron/proton delivery to the oxo-ferryl state (to avoid 7 2). Gerber and Sligar (42) have proposed a distal charge relay system to accomplish the proper delivery of protons to the iron-bound peroxide oxygen. The ``extra'' water in the active site during catalysis apparently provides an alternate source of protons to iron-bound peroxide, 6a and 6b, and the oxo-ferryl intermediate, 7, promoting water and/or hydrogen peroxide production. Removal of methyl groups from the substrate (as in norcamphor) or in mutant P450-CAM enzymes where active site valines are replaced with alanines (41) leads to uncoupling. Substrates like ethylbenzene and 1,1,1-trichloroethane(16, 20) , which certainly do not tightly fill the substrate binding pocket, also promote uncoupling. The results reported herein with 5-methylenylcamphor suggest that addition of a carbon atom to the camphor (the exocyclic olefin substituent at C-5) also leads to uncoupling.

Uncoupling also occurs when the potential substrate hydroxylation sites are blocked as has been observed in the present study with 5,5-difluorocamphor (Table 1). Ullrich and co-workers (43) examined the reaction of perfluoro-n-hexane with liver microsomal P450 and saw no product formation and a 2:1 NADPH/O(2) consumption ratio, due to the four-electron reduction of O(2) to H(2)O. Similar results are reported in this study for 5,5-difluorocamphor where there is very little formation of oxygenated product and a NADH/O(2) consumption ratio approaching 2:1. Other laboratories have previously reported a four-electron oxidase activity for mammalian and bacterial P450s(8, 25) . Loida and Sligar (16) have been able to vary the amount of two-electron and four-electron uncoupling observed in the benzylic hydroxylation of ethylbenzene through changes in the side chains of particular amino acids in the substrate binding pocket. The results herein indicate that the mode of uncoupling can be varied using different 5-substituted camphor analogues.

Replacement of the hydrogens on C-9 of 5,5-difluorocamphor with deuteriums increases the extent of four-electron uncoupling (Table 1). Analysis of the consumption of NADH by 5,5-difluorocamphor and its d(3) derivative under oxygen limiting conditions (Table 5) reveals significant differences in the distribution of alcohol and water products resulting from oxygen transfer from the reduction of intermediate 7, respectively. Deuteration of C-9 diminishes the formation of C-9 alcohol from 28.3 to 2.4% with a concomitant increase in the water oxidation reaction from 69.3 to 95.2%. Clearly, the fate of the oxo-oxygen atom in intermediate 7 is very sensitive to the presence of deuteriums at C-9 in 5,5-difluorocamphor. Interestingly, the extent of alcohol formation at C-6 is not affected by deuteration at C-9.



In summary, the oxygenation of camphor analogues in which the normal site of reaction, C-5, is either blocked or modified occurs with decreases in the rate of NADH consumption and with considerable uncoupling of electron and oxygen transfer. (1R)-5-Methylenylcamphor has been shown to undergo two-electron uncoupling wherein the decrease in oxygenated product is compensated for by the production of H(2)O(2). (1S)-5-Methylenylcamphor exhibits slow turnover but is only slightly uncoupled. Both enantiomers of 5,5-difluorocamphor and (1R)-9,9,9-d(3)-5,5-difluorocamphor are four-electron oxidase uncouplers. They presumably still go through the oxo-ferryl intermediate, 7 in Fig. 1, which is reduced to give a second molecule of water. The slowdown in the rate of oxygenated product formation (Table 3) and the intermolecular isotope effect of greater than 11 for reaction at C-9 of 5,5-difluorocamphor (Table 4) suggests that the rate-limiting step in the P450 cycle may have changed from electron transfer to oxygen transfer with 5,5-difluorocamphor.


FOOTNOTES

*
This research was supported by Research Corporation Grant R-128 (to T. A. B.), American Cancer Society Institutional Research Grant 107 (to T. A. B. and J. H. D.), and National Science Foundation Grant DMB-8605876 (to J. H. D.). Preliminary accounts of this research have been presented at the Second International Symposium on Cytochrome P450 of Microorganisms and Plants, Tokyo, Japan, June, 1993, and at the Eighth International Conference on Cytochrome P450: Biochemistry, Biophysics and Molecular Biology, Lisbon, Portugal, October, 1993. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry. Tel.: 803-777-7234; Fax: 803-777-9521; Dawson@chem.scarolina.edu.

(^1)
The abbreviations used were: P450, cytochrome P450; P450-CAM, the camphor-hydroxylating P450 from Pseudomonas putida; PdR, putidaredoxin reductase; Pd, putidaredoxin.


ACKNOWLEDGEMENTS

We are grateful to Mark P. Roach and to Drs. Masanori Sono, Barton K. Hawkins, Leonard M. Hjelmeland, Michael Walla, David P. Ballou, and Ronald E. White for helpful discussions and to Issa S. Isaac, William E. Kemnitzer, and Dr. David D. Peters for technical assistance.


REFERENCES

  1. Hawkins, B. K., and Dawson, J. H. (1992) Front. Biotransformation 7, 216-278
  2. Dawson, J. H., and Sono, M. (1987) Chem. Rev. 87, 1255-1276
  3. Dawson, J. H. (1988) Science 240, 433-439 [Medline] [Order article via Infotrieve]
  4. Sligar, S. G., and Murray, R. I. (1986) in Cytochrome P450: Structure, Mechanism and Biochemistry (Ortiz de Montellano, P. R., ed) pp. 443-479, Plenum Press, New York
  5. Gunsalus, I. C., Meeks, J. R., Lipscomb, I. D., Debrunner, P. G., and M ü nck, E. (1974) in Molecular Mechanisms of Oxygen Activation (Hayaishi, O., ed) pp. 559-613, Academic Press, New York
  6. Ortiz de Montellano, P. R. (1986) in Cytochrome P450: Structure, Mechanism and Biochemistry (Ortiz de Montellano, P. R., ed) pp. 216-271, Plenum Press, New York
  7. White, R. E. (1991) Pharmacol. Ther. 49, 21-42 [Medline] [Order article via Infotrieve]
  8. Atkins, W. M., and Sligar, S. G. (1987) J. Am. Chem. Soc. 109, 3754-3760
  9. Loida, P. J., Sligar, S. G., Paulsen, M. D., Arnold, G. E., and Ornstein, R. L. (1995) J. Biol. Chem. 270, 5326-5330 [Abstract/Free Full Text]
  10. Atkins, W. M., and Sligar, S. G. (1988) Biochemistry 27, 1610-1616 [Medline] [Order article via Infotrieve]
  11. Atkins, W. M., and Sligar, S. G. (1988) J. Biol. Chem. 263, 18842-18849 [Abstract/Free Full Text]
  12. White, R. E., McCarthy, M.-B., Egeberg, K. D., and Sligar, S. G. (1984) Arch. Biochem. Biophys. 228, 493-502 [Medline] [Order article via Infotrieve]
  13. Eble, K. S., and Dawson, J. H. (1984) J. Biol. Chem. 259, 14389-14393 [Abstract/Free Full Text]
  14. Gelb, M. H., Malkonen, P., and Sligar S. G. (1992) Biochem. Biophys. Res. Commun. 189, 488-495 [Medline] [Order article via Infotrieve]
  15. Maryniak, D. M., Kadkhodayan, S., Crull, G. B., Bryson, T. A., and Dawson, J. H. (1993) Tetrahedron 49, 9373-9384 [CrossRef]
  16. Loida, P. J., and Sligar, S. G. (1993) Biochemistry 32, 11530-11538 [Medline] [Order article via Infotrieve]
  17. Jones, J. P., Trager, W. F., and Carlson, T. J. (1993) J. Am. Chem. Soc. 115, 381-387
  18. Watanabe, Y., and Ishimura, Y. (1989) J. Am. Chem. Soc. 111, 410-411
  19. Fruetel, J. A., Collins, J. R., Camper, D. L., Loew, G. H., and Ortiz de Montellano, P. R. (1992) J. Am. Chem. Soc. 114, 6987-6993
  20. Lefever, M. R., and Wackett, L. P. (1994) Biochem. Biophys. Res. Commun. 201, 373-378 [CrossRef][Medline] [Order article via Infotrieve]
  21. Fruetel, J., Chang, Y.-T., Collins, J., Loew, G., and Ortiz de Montellano, P. R. (1994) J. Am. Chem. Soc. 116, 11643-11648
  22. De Voss, J. J., and Ortiz de Montellano, P. R. (1995) J. Am. Chem. Soc. 117, 4185-4186
  23. Brewer, C. B., and Peterson, J. A. (1988) J. Biol. Chem. 263, 791-798 [Abstract/Free Full Text]
  24. Groves, J. T. (1985) J. Chem. Ed. 62, 928-931
  25. Gorsky, L. D., Koop, D. R., and Coon, M. J. (1984) J. Biol. Chem. 259, 6812-6817 [Abstract/Free Full Text]
  26. O'Keeffe, D. H., Ebel, R. E., and Peterson, J. A. (1978) Methods Enzymol. 52, 151-157 [Medline] [Order article via Infotrieve]
  27. Kadkhodayan, S. (1992) Part I: Mechanistic Studies of Cytochrome P450-CAM with Different Camphor Analogues; Part II: Electronic Absorption and Magnetic Circular Dichroism Spectroscopy of Horseradish Peroxidase Reconstituted with Biomimetic Iron Porphyrin and Iron Chlorin Prosthetic Groups , Ph. D. thesis, University of South Carolina
  28. Gunsalus, I. C., and Wagner, G. C. (1978) Methods Enzymol. 52, 166-188 [Medline] [Order article via Infotrieve]
  29. Maryniak, D. M. (1992) Part I: Heteroatom Directed Diels-Alder Approach Towards CC-1065; Part II: Synthesis of Cytochrome P450-CAM Substrate Analogues , Ph. D. thesis, University of South Carolina
  30. Robinson, J., and Cooper, J. M. (1970) Anal. Biochem. 33, 390-399 [Medline] [Order article via Infotrieve]
  31. Poulos, T. L., Finzel, B. C., Gunsalus, I. C., Wagner, G. C., and Kraut, J. (1985) J. Biol. Chem. 260, 16122-16130 [Abstract/Free Full Text]
  32. Poulos, T. L., Finzel, B. C., and Howard, A. J. (1987) J. Mol. Biol. 195, 687-700 [Medline] [Order article via Infotrieve]
  33. Raag, R., and Poulos, T. L. (1989) Biochemistry 28, 917-922 [Medline] [Order article via Infotrieve]
  34. Gould, P. V., Gelb, M. H., and Sligar, S. G. (1981) J. Biol. Chem. 256, 6686-6691 [Abstract/Free Full Text]
  35. Ullah, A. J. H., Murray, R. I., Bhattacharyya, P. K., Wagner, G. C., and Gunsalus, I. C. (1990) J. Biol. Chem. 265, 1345-1351 [Abstract/Free Full Text]
  36. Crull, G. B., Kennington, J. W., Garber, A. R., Ellis, P. D., and Dawson, J. H. (1989) J. Biol. Chem. 264, 2649-2655 [Abstract/Free Full Text]
  37. Gelb, M. H., Heimbrook, D. C., and Sligar, S. G. (1982) Biochemistry 21, 370-377 [Medline] [Order article via Infotrieve]
  38. Hjelmeland, L. M., Aronow, L., and Trudell, J. R. (1977) Biochem. Biophys. Res. Commun. 76, 541-549
  39. Groves, J. T., McClusky, G. A., White, R. E., and Coon, M. J. (1978) Biochem. Biophys. Res. Commun. 81, 154-160 [Medline] [Order article via Infotrieve]
  40. Raag, R., and Poulos, T. L. (1991) Biochemistry 30, 2674-2684 [Medline] [Order article via Infotrieve]
  41. Atkins, W. M., and Sligar, S. G. (1987) J. Am. Chem. Soc. 111, 2715-2717
  42. Gerber, N. C., and Sligar, S. G. (1992) J. Am. Chem. Soc. 114, 8742-8743
  43. Staudt, H., Lichtenberger, F., and Ullrich, V. (1974) Eur. J. Biochem. 46, 99-106 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.