Scanning tunneling microscopy study of cytochrome P450 2B4 incorporated in proteoliposomes

Q SocEtk frangeise de biochimic et biologic moltcul~irc / Elscvier. Paris

VYu LTvarov”, YD Ivanova, BN Romanov”,

MO Gallyamovb,

01 Kiselyovab,

IV Yaminskyb

“Labrm~ror~vof Plt~sic.o-Ck~~tt~it~anl Methds of Analysis, Ittstitrcte of Bionwdiical Chemistty. Pogoditrskaya St IO. lIYX3~ Mos~I~M’: “Faculty r.$ Physics, Mosutw State Uttiver-sit?, Lenitzskie Gori. I19899 Mo.~~tw. Russia (Received 8 February 1996; accepted 30 September 1996)

Summary - la the present paper, the application of scanning tunneling microscopy in cytochrome P45Os membrane topology is discussed. The method enables visualization of heme location in the lipid-bilayer-incorporated protein. It is supposed that the membrane-bound cytochrome P4SO en t!ie tunneling microscope substrate should behave as ‘molecular diode’. A model explaining the liposome and the proteoliposome images observed is proposed. scanning tunneling microscopy / cytochromc P450 / proteoliposomes

Introduction

Results and discussion

Cytochromes P&O play an important role in the metabolism of many cndogenous and exogenous compounds and have long been the subject of active biochemical and medical research. These hemoproteins are represented by water-soluble and membrane-soluble isoforms. While the problems connected with the structures and functions of the first group of P4SOs are largely solved owing to X-ray studies I I-
We observed that with using chrome and nickel as supporting media, the unevenness of the layered relief was as low as about 200 .&, this value being of the same order as the ultrasonic liposome size. If gold coating was used, the needle-effected shift of liposomes over the surface was sometimes observed and in some cases the liposome destruction even occurred. Therefore, the use of these materials as supports impeded the topographic studies of the protein on Ihc liposome surface. We ascertained that liposome images on the surface of highly orientated pyrolytic grafite were invariably reproduced and the resolution of the liposome images obtained was much higher. This determined the choice of the highly orientated pyrolytic graphite’s surface as support to obtain protein images on the proteoliposome surface. Figure I presents images of liposomes and proteoliposomes on highly oriented pyrolytic graphite at the positive and negative polarity of tip potential (U = C 100 my). X, Y, image sizes; Z, apparent height along the vertical axis (gray color shades (from black to white) correspond to Z values growth). Use of such supporting substrate made possible stable imaging of objects. At the same time, with tne use of gold-, nickel- or chrome-coated substrates, reproduction of proteoliposome images, in different experimental series, proved to be instable. It can be seen that the average liposomal size on the X-Y plane is about 200 A which corresponds to these vesicles’ average diameter as determined by electron microscopic technique. The liposome image height varies from 15 to 60 A. Apparently with drying the sample on the substrate, the vesicles collapse. As shown in figure Ic, d, white or dark spots depending on tip potential polarity - show up on the proteoliposome surface. Of interest is the fact that they do

Materials and methods Cytochromc P4SO was prepared from rat liver microsomes [IO]. Ultrasonic liposomes and proteoliposomes were prepared from egg phosphatidylcholine as described in [ 1I]. The sample was placed onto the substrate and dried up. The measurements were taken in a constant current mode using the scanning tunneling microscope described earlier [ 121. Tips were prepared from Pt/lr wire. As supporting substrate, freshly cleaved highly oriented pyrolytic graphite was used. Heme degradation in cytochrome P450 2B4 was carried out by use of hydrogen oxide according to the earlier described procedure [ 131.

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Fig 1. STM images of liposomes (A, B) and proteoliposomes (C, D) at the positive (+) and negative (-) tip polarity. Cytochrome P450 molecules are denoted by arrows.

782 not cluster. To elucidate the nature of these spots, the crosssectioning of the obtained images was undertaken. As shown in figure 2, the liposomes' images were independent of tip potential polarity; As regards proteoliposomes, there were several pits (4-5 A deep) on their surface at the positive polarity, or some protrusions (7-8/~, high), at the negative one. On reverse switch, the image was fully restored. We suggest that it is the heme (alone or with the heme-surrounding area) that is responsible for the appearance of pits or protrusions, depending on tip potential polarity. To verify this suggestion, analogous experiments were carried out with proteoliposomes containing apocytochrome P450 2B4. It was found that the picture obtained with such proteoliposomes is similar to that seen with liposomes (the image of proteoliposomes with the apoenzyme is not shown because the picture obtained is essentially the same as the liposome-imaging one). The cross-section of the images was independent of tip potential polarity. Neither pits nor protrusions were seen on the vesicle surface, in full agreement with our assumption that the visualization of exactly P450 2B4s heme was attained. STM images may be ambiguous and uneven fragments of the support may be taken for liposome images. We assumed therefore that proteoliposome images' contrasts would be different for different tunnel current and shift values as was indeed observed in our experiments. Thus the proteoliposome images obtained were not artifacts. Special precautions were performed to minimize thermal drift, electronic and seismic noise of the STM. The average noise level was typically less than 0.02

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nm peak-to-peak on layered materials (HOPG, halcogenites etc) and metallic substrates (gold, silver, nickel, etc). The noise measurements have been described in detail [14]. We have observed some increase of the tunnel current noise on the surface of liposomes and liposome/protein complexes. However, the average noise level on the topography images does not exceed the value of 0.1 nm peak-to-peak. At this noise level the probability of the topography fluctuation, 0.7-1.5 nm in height and 1-3 nm in width, is less than a few percent for a single image. Reversible protrusions and pits were observed in the topography images in a set of experiments. The overall probability for the observation of the same fluctuation in set of N measurements is equal to V = ViN, where Vi is the probability for the observation of the topography fluctuation at a single image. This means that even for N = 3-4 the probability for the misinterpretation (regarding the fluctuation as a topography structure) is really diminishing. It is worth noting that no image processing which can lead to the broadening of the individual peaks (for example averaging) was performed. The slope reduction which is performed for better image presentation does not affect the height and width of individual peaks. Our statement that what we have observed is actually the heme area is confirmed by the following facts: - 1) When the heme is removed from cytochrome P450 ( as p,.Led in our paper) and cytochrome P450 is subsequently incorporated into the liposome, then [he liposome image does r,,ot show protrusions at negative tip polarity potential and hzts no pits at positive polarity. Tlae proteoliposome

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Fig 2. Cross-section of STM images of proteoliposomes (A, B) an.d approximation of liposome profile (C) at the positive (+) and negative (-) tip polarity.

783 image thus obtained is smooth and its form remains unchanged even with polarity changes. - 2) The liposome image without protein incorporated into liposome is not dependent on polarity and has no protrusions at negative tip polarity petential and no pits at positive polarity. The image is also smooth and its forms remains unchanged with tip polarity changes. - 3) The image of heme containing cytochrome P450 has some changes on liposome surface; one can see protrusions at negative tip polarity potential and pits at positive polarity. Thus it is the presence of heme that leads to the appearance of protrusions and pits on the proteoliposome image; hence this is not an artifact. To explain the changes in the proteoliposome image form, let us imagine the tunnel current as two succe~,s[ve processes of electron transport from the tip to the heine and then from the heine to the substrate. Let us imagine further the profile of the multi-dimensionai potential surface of reactants plus the environment (electron on the tip-protein-substrate) in nuclear coordinates as potential surface R (fig 3), in the same way as described in [15]. The profile of producrs potential surface plus the environment (tip-electron on heme-substrate) is reFesented in figure 3 as potential surface PI and products plus the environment (tip-heine-electron on substrate) as P2. In the study of Marcus and Sutin it was noted that the electron transport from state R to state Pi will be observed for those nuclear coordinates at which the intersection of potential curves R and Pi occurs [15]. We assume that in our experimental system distance rl (tip-heine) is much less than distance r2 (heine-support). As the electron transport rate constant K is directly proportional to exp (-b'r), where b is the constant of the reacting system, and r the distance between donor and acceptor, the equilibrium in the tip-heine system is set up mo=e rapidly than in the heine-support system. It may be presumed therefore that in the R---~PI----~P~_system the local equilibrium, R~---)PI is indeed realized. The probability W of an electron being located on the heine (ie when the heine iron's oxidation level equals +2) may be determined from the distribution: W = exp (-AGI/RT)/[ 1+ exp (-AGI/RT)]

( 1)

where AG~ is free energy of the electron transport reaction R--)Pi and T is the tempeiature. The electron transport reaction rate in the R-->PI-->Pz system is determined by its limiting state, ie by the rate of the process PI--)P2. Thus current ! in the R-->PI-->P2 system may be represented as: I = e*[W*Ki

- (I-W)*K2]

where e is the electron charge, K I is the rate constant of the process P~---)P2 and K2 is the rate constant of the process P2~PI. Taking into account that [ 15]: KI = B*exp[- (AG2+~)2/(4~,RT)]

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Fig 3. Profile of the multi-dimensional potential surface of reactants R and products PI, P2 of the reaction of electron transport from the STM tip onto the heine and from the heme onto the graphite substrate. For symbols R, Pl and P2 see the text.

K2 = Kl*exp (AG2/RT), where B is the constant for the given system, AG~, AG2standard free energy of the R~P~ and PI-->P2 reactions (AGI > 0, DG2 < 0 in our case) and ~, is a reorganizational term, we obtain: 1 = e* Kl* [W- (l-W) *exp (AG2/RT)] = e'B* exp[- (AG2+~.)-'/(4~.RT)] * [W- (l-W) * exp (AG2/RT)]2)t Substituting W, as in equation (1), into (2), we obtain: 1 = e (B*exp[- (-AG2+3.)2/(43.RT)] (exp (AGz/RT) (3) * {exp[- (AGI + AGz)/RT]-I }/[l+exp (-AGdRT)] where AGI+AG2 = U*e*NA, U the potential on tip and NA the Avogadro number. By way of discussion, it would be of interest to study the theoretical behavior of the dependence I = I (AU) characteristic of tunnel current I. Clearly, at AGI = AGE, ie with AU = 0, I = 0; at U < 0, ie with a high negative potential on the tip W = l, the current reaches its saturation I = Imax. = e* K1. At U > 0, ie with a high positive potential on the tip W = 0, the current reaches its saturation I = Imax*exp (AGE/RT). From this it follows that at the negative polarity the saturation current on the tip is exp ( lAG2 I/RT) times higher than at the positive one. Therefore with such electrical chain configuration, the enzyme should behave as 'molecular diode'. Thus, along with some other electron transport enzymes, such as mitochondrial succinate dehydrogenase [ 16], cytochrome P450 is also characterized by diode-like behavior. This will allow cytochrome P450 to find its appropriate place in the bioelectronics

784

Conclusions In the paper presented, the application of STM in topographic studies of cytochrome P450 in proteoliposomes is considered. The method proposed enables the imaging of the protein's heme in a proteoliposome. Random distribution of the protein over the proteoliposome surface ~ in the form of single objects, not clusters ~ ~ observed. A model describing the peculiarities of proteoliposomes' visu:'lization is proposed.

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