On the role of phospholipids in the cytochrome P450 enzyme system

The cytochrome P450 enzyme system is involved in the metabolism and elimination of an almost unlimited number of endogenous and exogenous substrates. Biotransformation by cytochromes P450 plays a role in the conversion xenobiotics into more hydrophilic products. Generally, this process of biotransformation in which cytochrome P450 reactions take part, leads to elimination of the xenobiotic through urine and / or faeces although in some cases this process can also lead to the formation of more toxic metabolites. Because of its significant role in the conversion of numerous endogenous and exogenous compounds, a complete understanding of the cytochrome P450 enzyme system is of importance in toxicology, pharmacology, anesthesiology, pathology and other related biomedical fields.Twenty-five years ago the phospholipids of the membrane of the endoplasmatic reticulum, to which the enzyme system is bound, appeared to play an important role in the in vitro cytochrome P450 enzyme system. Although the role of the membrane(phospholipids) in the cytochrome P450 system has been intensively studied since then, it has not resulted in a unanimous conclusion. The goal of this thesis was therefore, to gain further insight into the role of the membrane and membrane phospholipids in the cytochrome P450 system. Attention is thereby not only paid to the effects of the membrane and the phospholipids on cytochrome P450 enzymes but also to the effects on another important protein component of the enzyme system, namely NADPH-cytochrome reductase.In the first two chapters the cytochrome P450 enzyme system and the membrane(phospholipids) of the endoplasmatic reticulum are described respectively. In chapter 1A the structural and catalytical properties of the cytochrome P450 enzyme system are briefly discussed. Special attention is paid to the occurence, multiplicity, induction, the structure of the individual protein components of the enzyme system and the catalytic cycle of cytochromes P450. The individual steps in this catalytic cycle are discussed in detail.Chapter 1B deals with the structural aspects of the membrane phospholipids and the membrane of the endoplasmatic reticulum. In addition a brief description of the different types of reconstituted systems used in this thesis is given. Finally, the effects of phospholipids on the cytochrome P450 enzyme system, reported up to now in the literature, and various current hypotheses for the stimulating effect of phospholipids are discussed briefly.In chapter 2 results are described that characterise the sensitivity of microsomal and isolated cytochrome P450 IA1 and IIB1 for an organic hydroperoxide; cumene hydroperoxide (CuOOH). These data provide information on the difference in the way of membrane incorporation of these two cytochrome P450 enzymes. Up to now, very little attention has been paid to possible differences in sensitivity of cytochromes P450 to conditions of oxidative stress. A difference in sensitivity for (hydro)peroxides between different forms of cytochrome P450, as demonstrated with CuOOH in the present study, can be of importance from a toxicological point of view. Especially in cases in which conversion by one cytochrome P450 enzyme results in detoxification of the substrate whereas another cytochrome P450 enzyme causes bioactivation of the substrate, a difference in sensitivity for conditions of oxidative stress can then result in a shift in the metabolite pattern.Cytochrome P45 0 IIB1, embedded in the microsomal membrane is more sensitive towards CuOOH treatment than microsomal cytochrome P450 IA1. Purification of these enzymes and reconstitution in a system in which the proteins remain soluble results in a disappearance of the difference in CuOOH sensitivity between the two cytochrome P450 enzymes. Upon incorporation of cytochrome P450 IA1 and IIB1 into an artificial membrane, cytochrome P450 IIB1 again appears to be more sensitive towards CuOOH than cytochrome P450 IA1. Furthermore, the EC-50 values (effective cumene hydroperoxide concentration which causes 50% inhibition of cytochrome P450 dependent activities) in the microsomal and membrane incorporated reconstituted systems are comparable. Based on these results it is concluded that (1) the difference in sensitivity between cytochrome P450 IA1 and IIB1 towards treatment with CuOOH originates from a difference in the way these cytochrome P450 enzymes are incorporated into the membrane, that (2) the purification procedure does not affect the parameters determining the way of incorporation of the protein into the membrane and that (3) the way of membrane incorporation of cytochrome P450 enzymes in microsomal and reconstituted systems is comparable.In chapter 3 the effects of changes in the fatty acyl moiety of phosphatidylcholine (PC) from dilauroyl (di 12:0) to distearoyl (dil8:0) on the kinetics of reconstituted cytochrome P450 IA1 and IIB1 were investigated. So far, studies on the effect of phospholipids on the kinetics of cytochrome P450 dependent reactions have focussed on one P450 enzyme or one phospholipid. Furthermore, mainly the effect on the activity at non-saturating substrate concentrations was investigated without paying much attention to the effect on the kinetic parameters K m and V max , of a cytochrome P450 catalysed reaction. An effect of phospholipids on for example the K m could be of considerable importance especially because in living organism the substrate concentrations will generally be low. Furthermore, a different effect of phospholipids on the substrate's apparent Km of different cytochrome P450 enzymes - converting the substrate to different metabolites - might not only affect substrate conversion rates but also the metabolite pattern.The results presented in chapter 3 demonstrate that the V max of the cytochrome P450 dependent O-dealkylation of alkoxyresorufins and ethoxycoumarin for both cytochrome P450 IA1 and IIB1 is two times higher in the PC di 12:0 system compared to the PC di 18:0 system. The effect of a change in the fatty acyl moieties on the Km of the xenobiotic substrate however, appeared to be different for the two cytochrome P450 enzymes. For cytochrome P450 IA1 the K m appeared to be lower in the PC di 12:0 system compared to the PC dil8:0 system whereas for cytochrome P450 IIB1 the K m was lowest in the PC di l8:0 system. Additional results demonstrated that the kinetic parameters were dependent on the PC : P450 ratio and that changing this ratio affected the kinetic parameters of cytochrome P450 IA1 and IIB1 in a different way.The reason for the differential effect on the substrate apparent K m was further investigated in a series of experiments in which the effect of PC di 12:0 and PC di l8:0 on individual steps of the catalytic cycle of cytochrome P450, like substrate binding, oxygen binding and rate of electron transfer, was studied. From these experiments it was concluded that the higher V max in the PC di 12:0 system, observed for both cytochrome P450 enzymes, was at least in part due to the higher affinity of cytochrome P450 for NADPH-cytochrome reductase in the PC di 12:0 system. Furthermore, these experiments demonstrated that the different effect of a change in the fatty acyl moieties of PC on the K m of cytochrome P450 IA1 and IIB1 did not result from a different effect on substrate binding, oxygen binding and rate of electron transfer. This means that the differential effect on the K m must result from an effect on one or more of the other steps in the catalytic cycle such as reductive oxygen splitting, substrate conversion and / or product release.The results show that the effect of a change in the type of PC and or the PC : P450 ratio on the kinetic parameters, K m and V max , is dependent on the cytochrome P450 enzyme used in the reconstitution. Furthermore, in contrast to what is generally assumed and based on results under non- saturating substrate conditions [1 -4], the addition of PC appears to result for some cytochrome P450 enzymes in a decrease of the V max in the reconstituted system. The results in chapter 3 also demonstrate that this is not reflected in lower but - in contrast - in higher conversion rates at non- saturating substrate concentrations (Figure 9.1), because the K m is decreased simultaneously.In chapter 4 the existence of a preference - with respect to binding - of cytochrome P450 IIB1 for phospholipids with certain headgroups or fatty acyl moieties was investigated. The existence of "boundary" phospholipids (phospholipids which bind to cytochrome P450 with specificity and high affinity) for microsomal cytochrome P450 has been a topic for several studies. Nevertheless, little is known about this subject and unanimous conclusions have not been reached. It has been suggested that the composition of the membrane in the direct vicinity of cytochromes P450 is different from the rest of the membrane [5] and that specific interactions exist between cytochrome P450 and phosphatidylethanolamine (PE) [61 and phosphatidic acid (PA) [7].The results in chapter 4 show that the apparent binding constant (K d ) of a cytochrome P-450 IIB1 - phospholipid complex is dependent on the degree of unsaturation of the phospholipid side chains; demonstrating a decrease in the Kd with increasing degree of unsaturation, but independent of the length of the acyl chains. In addition, the apparent Kd appeared to be dependent on the headgroup of the phospholipid molecule, showing a significantly higher K d for PE di 16:0 compared to PC di 16:0, PS di 16:0 and PI 16:0/18:1.Translation of these results to the in vivo situation has to be done with caution because the results were obtained in a reconstituted system with isolated, solubilised cytochrome P450. In the membrane of the endoplasmatic reticulum other factors such as for example the presence of other proteins can play an additional important role in the interaction of cytochrome P450 with phospholipids. Furthermore, the effect of the length and the degree of unsaturation of the fatty acyl chains on the Kd was determined for PC. It remains to be established whether for phospholipids with different headgroups similar influences of the length an degree of unsaturation of the acyl chains are observed. Investigations in this direction are however, seriously hampered by the fact that series of pure molecular species of PE, PS and PI are not commercially available.In chapter 5 the existence of specific phospholipid : protein interactions for NADPH -cytochrome reductase was investigated. Compared to cytochrome P450 very little attention has been paid to possible interactions between phospholipids and NADPH-cytochrome reductase and possible consequences of such interactions for the cytochrome P450 system. NADPH-cytochrome reductase is a very important component of the cytochrome P450 enzyme system and the stimulating effect of phospholipids on the rate of cytochrome P450 dependent reactions may in part originate from an effect on NADPH-cytochrome reductase resulting in a more efficient electron transfer to the cytochromes P450.Based on the results from 31P-NMR experiments and chemical analysis, it was concluded that NADPH-cytochrome reductase exhibits a preference for the negatively charged phospholipids phosphatidylserine (PS) and phosphatidylinositol (PI). In addition, experiments investigating the possible consequences of a special interaction of NADPH-cytochrome reductase with PS and PI demonstrated that (1) PS and PI had a significantly different effect on the DPH-PC dependent quenching of tryptophan fluorescence of NADPH-cytochrome redcutase compared to PE and PC and that (2) the V max of cytochrome P450 IIB1 dependent O-dealkylation of pentoxyresorufin in the presence of 1:1 mixtures of PS:PC and PI:PC were respectively higher and lower compared to the Vmax in the presence of a 1:1 mixture of PE:PC or PC alone. These phenomena might best be explained by a PS and PI induced specific change in the conformation of NADPH-cytochrome reductase. Regarding the fact that the specific interaction in both cases involves a negatively charged phospholipid suggest a possible role of the phospholipid charge. However, the fact that the effects of PS and PI on the V max of the cytochrome P450 catalysed reaction are different demonstrates that phospholipid charge cannot be the only factor.In chapter 6 the redox cycling of 7-alkoxyresorufins and the product of their metabolism by cytochrome P450, resorufin, by NADPH-cytochrome reductase is investigated. Redox cycling is a process in which a substrate is I-electron reduced, in this case by NADPH-cytochrome reductase. The electron is transfered to molecular oxygen and the substrate is returned to its initial state and can enter a new cycle. During this process reactive oxygen species are formed which can initiate lipidperoxidation and / or inactivate cytochrome P450. Especially in systems in which the NADPH-cytochrome reductase concentration is relatively high this process is a disturbing side-reaction, because it uses up reduction equivalents resulting only in the formation of reactive oxygen species which may cause protein inactivation and lipidperoxidation. In the reconstituted systems used in this thesis redox cycling can play an important role because the NADPH-cytochrome reductase : cytochrome P450 ratio is 15 to 30 times higher than in the in vivo situation. Furthermore, cytochrome P450 has been demonstrated to be very sensitive to lipidhydroperoxides - formed during lipidperoxidation - and reactive oxygen species.The results of the present chapter demonstrate that at physiological pH alkoxyresorufins are much better substrates for redox cycling than resorufin. The inability of resorufin to stimulate redox cycling originates from the fact that at physiological pH resorufin exists mainly in its deprotonated form and this form is a much worse substrate for redox cycling than its protonated form. AM1 molecular orbital computer calculations demonstrated that the energy (E) of the lowest unoccupied molecular orbital (LUMO), i.e. the orbital into which the electron will be placed during redox cycling, of the deprotonated form is higher compared to the E LUMO of the protonated form. Furhermore, one-electron reduction of the protonated form appeared to be energetically favorable by 363.5 kJ/mol over one-electron reduction of the deprotonated form. In addition, the computer calculations demonstrated that the one electron reduced resorufin is most likely to become protonated at the O-atom of the intramolecular semiquinone imine moiety before reduction by a second electron. Finally, it was demonstrated that incorporation of NADPH-cytochrome reductase into an artificial membrane results in an increased redox cycling activity of resorufin compared to solubilized NADPH-cytochrome reductase. This was explained by an increase in the protonated form in the membrane either by (1) favored partitioning of the protonated form into the membrane or by (2) an effect of the membrane on the protonation equilibrium of resorufin in favor of the protonated form. This result points at the role of the membrane in concentrating apolar substrates of the cytochroom P450 : NADPH-cytochrome reductase svstem [8-10].In chapter 7 the use of AM1 MO calculations in predicting the ability of compounds to stimulate redox cycling, as demonstrated in chapter 6, was further investigated. Therefore, in addition to resorufins, the redox cycling ability of 1,4-benzoquinones was investigated. Quinones are toxic compounds that are often used in chemistry. They are also used in pharmacology, for example as anticancer drugs because of their toxic character. The mechanism through which quinones exert their toxic effects is believed to involve the covalent binding of quinones to cellular nucleophilic macromolecules and /or the quinone catalysed process of redox cycling. 1,4- Benzoquinone has been demonstrated to redox cycle very poorly. In the literature, the poor redox cycling of 1,4-benzoquinone has been ascribed to a very high 1-electron reduction potential. The results from chapter 7 demonstrate however, that, at physiological pH, 1,4-benzoquinone is quickly 2-electron reduced by NADPH-cytochrome reductase to form 1,4- hydroquinone. Instead of transfering its electron on to molecular oxygen, the 1 -electron reduced semiquinone is protonated and subsequently reduced by a second electron. However, at pH>9 the 1,4-benzoquinone appears to be capable of stimulating redox cycling. Furthermore, the pH- and concentration-dependencies of redox cycling in a system with NADPH- cytochrome reductase between 1,4-benzoquinone and 1,4-hydroquinone are demonstrated to be similar. Based on these observations it was concluded that 1,4-benzoquinone is capable of redox cycling from its deprotonated, 2-electron reduced form at relatively high pH levels which ensures an adequate concentration of the deprotonated form The results from chapters 6 and 7 demonstrate the importance of the protonation / deprotonation equilibrium of the I- and 2-electron reduced forms in the redox cycling process.In the paragraphs above the results of the study on the role of phospholipids in the cytochrome P450 : NADPH-cytochrome reductase system as it was executed for this PhD thesis are presented. Ever since 1968 [11] the role of phospholipids in this system has been a topic for numerous studies in which many questions concerning this role have been answered but many new questions have also been raised. Unfortunately, this thesis does not provide the answers to all these new questions because the cytochrome P450 system is too complex and the number of different phospholipids and the effects they induce is too large. Answering these questions will require many, many years of additional research. The goal of this thesis was to investigate certain aspects of phospholipids and the cytochrome P450 enzyme system to which up to now little attention has been paid in order to gain further insight into the role of phospholipids in the cytochrome P450 enzyme system.Altogether, the results of the experiments in this thesis present some new insightsin the role of phospholipids on the cytochrome P450 enzyme system. Furthermore, additional evidence for already existing hypotheses of the stimulating effect of phospholipids are also presented. The conclusions of the present thesis can be summarized as follows.(1) Cytochromes P450 can differ in the way they are incorporated in the membrane which might result in differences in their sensitivity towards cumene hydroperoxide.(2) The way of incorporation of cytochrome P450 enzymes in microsomal and reconstituted systems is comparable.(3) Phospholipids influence the kinetic parameters, Km and Vmax, of cytochrome P450 catalysed reactions in reconstituted systems.(4) The result of the effect phospholipids on the kinetic parameters of cytochrome P450 dependent reactions is not the same for all P450 enzymes.(5) The affinity of cytochrome P450 for NADPH-cytochrome reductase in a reconstituted system is dependent on the fatty acyl moiety of the phospholipid added to the system.(6) Phospholipids can decrease the apparent Kd of cytochromes P450 for their xenobiotic substrates.(7) The apparent K d of cytochromes P450 for phospholipids is dependent on the headgroup of the phospholipid and the degree of unsaturation of the fatty acyl chains but independent of the length of the acyl chains of the PC molecule.(8) There is a specific interaction between NADPH-cytochrome reductase and the negatively charged phospholipids PS and PI.(9) The membrane functions as a place where apolar substrates can accumulate thereby decreasing the apparent K m .(10) The membrane causes a shift in the overall protonation equilibrium of the substrate towards the protonated form.In addition to these conclusions a number of other interesting phenomena have been observed that have no bearing on the role of phopsholipids in the cytochrome P450 enzyme system but are also worth mentioning again.(1) The parameters determining the way of incorporation of cytochromes P450 in the membrane are not affected by the isolation procedure.(2) AM1 molecular orbital calculations is a useful additional tool in investigating the redox cycling capacity of chemicals.(3) The protonationequilibria of I- and 2-electron reduced compounds play an important role in their redox cycling ability.Finally, regarding the results of the experiments presented in this thesis one final conclusion must be added. The effect of phospholipids on the cytochrome P450 enzyme system is dependent on many factors such as the cytochrome P450 form, the fatty acyl moiety and headgroup of the phospholipid, the P450 : reductase ratio and the phospholipid : P450 ratio. Therefore, for a complete understanding of the mechanism(s) of action of phospholipids in the cytochrome P450 enzyme system, a detailed investigation of the effects of all phospholipids (and mixtures of phospholipids) on all P-450 forms at several P-450 : reductase and phospholipid : P-450 ratio's is necessary. This requires a vast amount of work although some of this work has already been done in these last twent