Vectorially-Oriented Monolayers of Cytochrome Oxidase: Fabrication

Mar 19, 1997 - Patterson function analysis of meridional X-ray diffraction from the LB-deposited monolayers has shown the profile structure of the so-...
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Vectorially-Oriented Monolayers of Cytochrome Oxidase: Fabrication and Profile Structures Ann M. Edwards,* Janine A. Chupa,† Robert M. Strongin,‡ Amos B. Smith, III, and J. Kent Blasie Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104

John C. Bean Bell Laboratories/Lucent Technologies, Murray Hill, New Jersey 07974 Received August 5, 1996X Vectorially-oriented monolayers of detergent-solubilized bovine heart cytochrome c oxidase have been formed by self-assembly from solution and Langmuir-Blodgett (LB) deposition. Both quartz and Ge/Si multilayer substrates, the latter fabricated by molecular beam epitaxy, were alkylated with an amineterminated alkylsiloxane monolayer prior to introduction to the protein. For the self-assembled protein monolayers, the amine end group surface provided for primarily electrostatic interactions with the protein, thereby encouraging a nearly unidirectional vectorial orientation of the so-adsorbed integral membrane protein. This was demonstrated by the analysis of meridional X-ray diffraction data from the monolayers so-adsorbed onto the Ge/Si multilayer substrates, which directly provided electron density profiles of the protein along the axis normal to the substrate plane to a spatial resolution of 10 Å. These profiles are consistent with the three-dimensional structure of the protein, obtained from electron microscopy. Patterson function analysis of meridional X-ray diffraction from the LB-deposited monolayers has shown the profile structure of the so-deposited protein monolayers to be qualitatively similar to that obtained via selfassembly from solution, thereby suggesting that the LB-deposited monolayers are similarly vectoriallyoriented. Optical spectroscopy using quartz substrates has also indicated that the LB monolayers tend to be more densely packed than their self-assembled counterparts. Optical linear dichroism has confirmed that the planes of the oxidase’s two heme groups and, hence, the molecule’s long axis are more perpendicular to the monolayer plane in the LB case than for the self-assembled monolayers, consistent with the profile length of the molecule along the axis normal to the monolayer plane. Such densely packed, vectoriallyoriented monolayers in a fully hydrated state now provide a unique opportunity to perform directly correlated structural-functional studies on this membrane protein.

Introduction Single monolayers of fully-functional electron transport membrane proteins, or supramolecular complexes thereof, vectorially-oriented on the surface of solid substrates have the potential to provide definitive information concerning the mechanism of their biological electron and ion transport function. [The three-dimensional structure of a protein molecule at atomic resolution is, in general, asymmetric; hence, the orientation of the protein molecule in three-dimensional space can be represented by a single vector referenced to some convenient aspect of the protein’s physical-chemical structure within the internal coordinate frame of the molecule. For membrane proteins, “vectorial orientation” refers to the orientation of that vector representing the internal coordinate frame of the protein molecule relative to the coordinate frame of the host membrane, for example, either the plane of the membrane or the normal to that plane.] This potential derives from the possibility to both control the state of the protein redox centers (e.g., electrochemically) and simultaneously determine key features of the protein structure (e.g., relative positions/orientations of redox centers) for this particular form of these proteins. In addition, the designed fabrication of such monolayer systems allows the exploration of the utility of these proteins, or specif* Author to whom all correspondence should be addressed. † Current address: Colgate-Palmolive Co., 909 River Rd, Piscataway, NJ 08855. ‡ Current address: LSU Chemistry Department, Baton Rouge, LA 70803. X Abstract published in Advance ACS Abstracts, February 1, 1997.

S0743-7463(96)00769-X CCC: $14.00

ically designed models thereof,1,2 in tailoring the macroscopic electro-optical response of the substrate surface. We have demonstrated via structural studies the ability to vectorially orient both peripheral membrane proteins and detergent-solubilized integral membrane proteins at the soft interface between an aqueous medium and the end groups of an organic self-assembled monolayer (SAM) chemisorbed onto the surface of a solid substrate employing designed specific interactions between particular residues on the protein’s surface and the SAM’s end groups.3 While other research groups have focused on measurements of the functional aspects of such (or closely related, e.g., Langmuir-Blodgett) monolayer systems which were not structurally characterized,4-8 we have focused instead on developing initially the physical techniques essential to determining the key structural features of the proteins within such vectorially-oriented single monolayers. To date, this work has concerned the determination of the so-called profile structures of such (1) Robertson, D. E.; Farid, R. S.; Moser, C. C.; Urbauer, J. L.; Mulholland, S. E.; Pidikiti, R.; Lear, J. D.; Wand, A. J.; DeGrado, W. F.; Dutton, P. L. Nature 1994, 368, 425. (2) Rabanal, F.; Gibney, B. R.; DeGrado, W. F.; Moser, C. C.; Dutton, P. L. Inorg. Chim. Acta 1996, 243, 213. (3) Chupa, J. A.; McCauley, J. P., Jr.; Strongin, R. M.; Smith, A. B., III; Blasie, J. K.; Peticolas, L. J.; Bean, J. C. Biophys. J. 1994, 67, 336. (4) Cullinson, J. K.; Hawkridge F. M.; Nakashima, N.; Yoshikawa, S. Langmuir 1994, 10, 877. (5) Jiang, M.; Nolting, B.; Stayton, P. S.; Sligar, S. G. Langmuir 1996, 12, 1278. (6) Guo, L.-H.; McLendon, G.; Razafitrimo, H.; Gao, Y. J. Mater. Chem. 1996, 6, 369. (7) Song, S.; Clark, R. A.; Bowden, E. F.; Tarlov, M. J. J. Phys. Chem. 1993, 97, 6564. (8) Owaku, K.; Goto, M.; Ikariyama, Y.; Aizawa, M. Anal. Chem. 1995, 67, 1613.

© 1997 American Chemical Society

Vectorially-Oriented Cytochrome Oxidase Monolayers

monolayers uniquely and to a resolution of ∼7 Å by X-ray interferometry/holography3,9 which is essential to the verification of the monolayer assembly process, including the protein vectorial orientation, the positions of the protein metal redox centers within this profile structure by resonance X-ray diffraction to within (1-3 Å,10 and the orientations of the redox centers within the monolayer structure by polarized X-ray spectroscopy11 and optical linear dichroism.12 These techniques have now been successfully applied to vectorially-oriented monolayers of cytochrome c covalently tethered to thiol SAM end groups and detergent-solubilized photosynthetic reaction centers electrostatically tethered to amine SAM end groups:3 two membrane proteins whose atomic (or near-atomic) resolution structures were already determined by X-ray crystallography. In this work, we have extended this initially structural approach to single monolayers of mitochondrial cytochrome oxidase, whose 20 Å resolution three-dimensional structure was already determined by electron microscopy13 but whose near-atomic resolution structure via X-ray crystallography has only just recently been published.14 Our first results demonstrate that the detergent-solubilized cytochrome oxidase can be electrostatically tethered to amine SAM end groups and thereby vectorially-oriented at the soft interface, via either self-assembly from solution or Langmuir-Blodgett deposition, and that the long axis of the integral membrane protein is tilted relative to the monolayer normal dependent on the fabrication method. This work has been briefly reported elsewhere.15 Experimental Section The Ge/Si multilayer substrates utilized in the X-ray diffraction experiments were fabricated by molecular beam epitaxy (MBE) at Bell Laboratories. Four inch diameter, p-type Si(100) wafers (SEH-America, Vancouver, WA) with a resistivity of 20 Ω cm were smoothed with 30 atomic monolayers of silicon (i.e., Si30) prior to deposition of the two unit cell superlattice structure of the form 2(Ge2Si30). Details of the MBE deposition procedures are given elsewhere.16 The use of only two unit cells in this inorganic reference structure provides continuous meridional X-ray diffraction over a wide range of qz (the reciprocal space axis perpendicular to the substrate plane) and, hence, guarantees the maximum amount of interference with the scattering from the unknown organic/bio-organic overlayers to be attached later.17 Furthermore, the more electron-dense Ge layers of the MBE substrates were chosen to be very thin to provide considerably more intense X-ray diffraction out to larger qz than was previously obtained using substrates fabricated by magnetron sputtering.18,19 The wafers were cut with a diamond pencil to obtain 1 (9) Blasie, J. K.; Xu, S.; Murphy, M.; Chupa., J.; McCauley, J. P., Jr.; Smith, A. B., III; Peticolas, L. J.; Bean, J. C. Mater. Res. Soc. Symp. Proc. 1992, 237, 399. (10) Pachence, J. M.; Fischetti, R. F.; Blasie, J. K. Biophys. J. 1989, 56, 327. (11) Zhang, K.; Edwards, A. M.; Dong, J.; Chupa, J. A.; Blasie, J. K. Proceedimgs of the Ninth International Conference on X-ray Absorption Fine Structure, Grenoble, 1996. To be published in J. Phys. IV. (12) Pachence, J. M.; Amador, S.; Maniara, G.; Vanderkooi, J.; Dutton, P. L.; Blasie, J. K. Biophys. J. 1990, 58, 379. (13) Valpuesta, J. M.; Henderson, R.; Frey, T. G. J. Mol. Biol. 1990, 214, 237. (14) Tsukihara, T.; Aoyama, H.; Yamashita, E.; Tomizaki, T.; Yamaguchi, H.; Shizawa-Itoh, K.; Nakashima, R.; Yaono, R.; Yoshikawa, S. Science 1996, 272, 1136. (15) Edwards, A. M.; Chupa, J. A.; Strongin, R. M.; Smith, A. B., III; Blasie, J. K.; Peticolas, L. J.; Bean, J. C. Biophys. J. 1996, 70, A263 (meeting abstract TuPMG3). (16) Bean, J. C.; Feldman, L. C.; Fiory, A. T.; Nakahara, S.; Robinson, I. K. J. Vac. Sci. Technol. 1984, A2, 436. (17) Cowley, J. M. Diffraction Physics, 2nd revised ed.; North-Holland Publishing Co.: Amsterdam, 1981. (18) Amador, S. M.; Pachence, J. M.; Fischetti, R.; McCauley, J. P., Jr.; Smith, A. B., III; Blasie, J. K. Langmuir 1993, 9, 812. (19) Xu, S.; Murphy, M. A.; Amador, S. M.; Blasie, J. K. J. Phys. I 1991, 1, 1131.

Langmuir, Vol. 13, No. 6, 1997 1635 cm × 2 cm × 0.5 mm substrates. The 1 mm thick quartz substrates (Esco products, Oak Ridge, NJ) used in both the polarized and nonpolarized optical spectroscopy were also cut to 1 cm × 2 cm. The synthesis of the organic compound [11-[N-(tert-butoxycarbonyl)amino]undecyl]triethoxysilane has been described in detail previously.3 Self-assembled monolayers (SAMs) of this compound were formed on the surfaces of both quartz and Si/Ge multilayer substrates using the modified method of Sagiv20 as described by Xu21 with the further modifications of eliminating the 5 mM NaOH cleaning step and increasing the time that the substrates were sonicated in a solution containing the triethoxysilane compound from 22 to 45 min. The macroscopic wettability of all resulting “protected amine” SAMs was routinely tested to ensure that the entire alkylated surface was uniformly hydrophobic irrespective of the two types of substrate utilized. The protected amine SAMs were then either stored under argon or deprotected for immediate use by immersing the coated substrates in concentrated HCl for 1.5 h to cleave the protecting tert-butoxycarbonyl groups by acid hydrolysis and leave the amine-terminated SAM, 11-siloxyundecaneamine. After the resulting deprotected amine SAM was rinsed well in ultrapure water (Millipore Corp., Bedford, MA), the wettability of the SAM routinely found it to be hydrophilic over the entire alkylated surface irrespective of the two types of substrate utilized. The active amine surface of these deprotected SAMs was then ready for introduction to the protein, cytochrome oxidase. All solutions used in the preparation and study of cytochrome oxidase were made with ultrapure water unless otherwise stated. The integral membrane protein, cytochrome c oxidase, was isolated and purified from beef heart mitochondria using further modifications of the method of Yonetani et al.22,23 as provided in the Supporting Information (three pages outlining the detailed cytochrome oxidase isolation and purification procedure). While an ionic detergent (potassium cholate) was used to facilitate the salt precipitations during purification, the purified cytochrome oxidase pellets were dissolved in a minimum of the nonionic detergent 0.1% (wt/vol) n-dodecyl β-D-maltoside (Sigma Chemical Co., St. Louis, MO) in 1 mM KPO4, pH 7.7, in order to restore enzymatic activity to the cytochrome oxidase24,25 which is inhibited by cholate. The resulting solution was divided into aliquots and flash frozen for storage at -80 °C. To produce the self-assembled cytochrome oxidase monolayer samples, amine-terminated SAMs were incubated for 72-96 h in solutions of 5-15 µM cytochrome oxidase in 0.1% n-dodecyl β-D-maltoside in 1 mM KPO4, pH 7.4. Prior to study, each specimen was removed from the protein solution and rinsed repeatedly with a detergent buffer solution of 0.1% n-dodecyl β-D-maltoside in 1 mM KPO4, pH 7.4, until no further protein could be removed, as determined via optical absorption spectroscopy (see below). This rinsing procedure was employed to ensure the removal of nonspecifically bound protein. To form the Langmuir-Blodgett-deposited cytochrome oxidase monolayer samples, substrates with an amine-terminated SAM were translated vertically at 3 mm/min upward through a monolayer of the protein spread on the surface of a Lauda Langmuir trough system (Brinkman Instruments, Inc., Westbury, NY) filled with a subphase of 1 mM CdCl2 (MCB Manufacturing Chemists, Inc., Cincinnati, OH) in 1 mM HEPES (Sigma Chemical Co., St. Louis, MO), pH 7.4. The protein was spread at the air-water interface by placing a (qz)crit, the specular scattering, |Fspec(qz)|, approaches zero rapidly and monotonically and, thus, |Ftot[qz>(qz)crit]|2 f |Fkin(qz)|2. Therefore, the total meridional X-ray scattering data from these specimens, I(qz) for (qz)crit < (qz)min e qz e (qz)max, is dominated by the kinematical diffraction arising from the electron density contrast profile, ∆F(z). The Ge/Si multilayer substrate has very narrow, largeamplitude features in its electron density contrast profile, and its profile structure is essentially known from its fabrication specifications. The addition of the SAM or SAM/protein overlayers makes a relatively small contribution to the profile structure of the composite system, since their electron density contrast profiles are expected to contain broad, low-amplitude features as compared to those in the profile of the Ge/Si multilayer substrate. The known profile structure of the Ge/Si multilayer substrate can thus be used to determine the profile structure of the SAM or SAM/protein overlayers by X-ray interferometry,32 (31) Murphy, M.; Blasie, J. K.; Peticolas, L. J.; Bean, J. C. Langmuir 1993, 9, 1134. (32) Lesslauer, W.; Blasie, J. K. Acta Crystallogr. 1971, A27, 456.

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as described below. The kinematical meridional X-ray diffraction for the SAM/protein composite structure, as shown in Figure 2b, is given by eq 3

|Fkin(qz)|2 ) |Fk(qz)|2 + |Fu(qz)|2 + |Fk(qz)||Fu(qz)| cos{[Ψk(qz)-Ψu(qz)] + [2πqzAku]} (3) where |Fkin(qz)|2 is the total kinematical structure factor modulus squared of the composite structure and |Fk(qz)|2 and |Fu(qz)|2 are the kinematical structure factor moduli squared of the known multilayer substrate and the unknown SAM/protein overlayers, respectively. These three structure factor moduli could be obtained experimentally from the kinematical X-ray diffraction from the composite system, from the Ge/Si multilayer substrate itself, and from the SAM/enzyme overlayers on a uniform Si substrate. The terms Ψk and Ψu are the phases of their respective structure factors, each referenced to the center of mass of their respective profile structures. Since the reference profile structure of the Ge/Si multilayer substrate and, therefore, its structure factor are known, Ψk is known. The term Ψu is unknown. The term Aku is the distance along the z-axis between the centers of mass of the Ge/Si multilayer substrate and the SAM/enzyme overlayers. If the mathematical substitution Ψ′u ) [Ψu - 2πqzAku] is made, then the center of mass of the profile of the unknown overlayer structure is referenced to the center of mass of the profile of the known multilayer reference structure. Solving for the only remaining unknown, Ψ′u, allows determination of the unknown profile structure of the SAM/protein overlayer. The third term in eq 3 represents the critical interference between the strong kinematical diffraction from the Ge/Si multilayer substrate and the weak kinematical diffraction from the SAM or the SAM/protein overlayer. Its effect is apparent from the differences between the meridional X-ray diffraction data of the Ge/Si multilayer substrates (Figure 2a) and those of the substrates plus amine SAM/ protein shown in Figure 2b. Evidence of destructive interference is particularly dramatic in Figure 2 where the peak at qz ∼ 0.045 Å-1 in Figure 2a for the bare Ge/Si substrate becomes a split peak of greatly reduced amplitude in Figure 2b for the amine SAM/self-assembled cytochrome oxidase system. For comparison, the data are also shown as their corrected meridional intensity functions in Figure 3. The term |Fkin(qz)|2 for the bare Ge/Si multilayer substrate, the substrate plus protected amine SAM, and the composite multilayer substrate/amine SAM/protein systems was obtained from the Lorentz-corrected, meridional elastic X-ray scattering, Ic(qz), by subtraction of |Fspec(qz)|2, as approximated by the Lorentz-corrected meridional scattering from a uniform silicon substrate.21,31 A Lorentz factor of qz was applied to correct for the ω-oscillations of the specimens.33 This procedure resulted in the kinematical diffraction data being restricted to the qz range (qz)min ≈0.011 e qz e (qz)max ≈0.110 Å-1 for the self-assembled specimens. All Fourier analyses, via both interferometry and holography, were restricted to this qz window. X-ray interferometric analysis of the meridional kinematical X-ray diffraction data was performed utilizing a highly constrained real-space refinement algorithm as described previously19 to accomplish the interferometric phasing of these data. The procedure involved first (33) Skita, V.; Filipkowski, M.; Garito, A. F.; Blasie, J. K. Phys. Rev. B 1986, 34, 5826.

Figure 3. Corrected (see Results) meridional intensity functions, Ic(qz), for the data shown in Figure 2 for (a) a typical bare two unit cell Ge/Si multilayer substrate and (b) for the same substrate plus amine-terminated SAM with self-assembled cytochrome oxidase electrostatically tethered. Dashed lines have been added to help guide the eye for comparative purposes.

establishing the relative electron density profile for the “known” Ge/Si multilayer substrate, with the initial models for the substrate being constructed on an absolute electron density scale based on the fabrication specifications. These models were then initially relaxed via a model refinement analysis, by comparing the calculated structure factor modulus squared and its unique Fourier transform, the Patterson function, for the models with their corresponding experimental meridional diffraction data and Patterson functions, subject to the same qz window as the experimental meridional X-ray diffraction data. Once reasonable (i.e., close but not perfect) agreement had been achieved between the experimental functions and their model counterparts, the constrained real-space refinement algorithm was employed as a final relaxation procedure. The interior portion of the so-refined model relative electron density profile for the bare Ge/Si multilayer substrate (i.e., Ge2Si30Ge2) was utilized as the primary constraint. This procedure yielded the experimental relative electron density profile for the bare substrate, which exactly predicted both the experimental intensity and Patterson functions. Only the interior portion of this “known” relative electron density profile structure was then used as the reference structure for the constrained real-space refinement of the meridional X-ray diffraction for the multilayer substrate/protected amine SAM or the multilayer substrate/deprotected amine SAM/protein system because of the modification of the outer silicon layer of the substrate that occurs upon formation of the SAM on its surface by chemisorption.21 The highly constrained real-space refinement algorithm yields one solution to a finite number of possible solutions for the phase of the kinematical structure factor, using the phase dominance of the known reference structure to force the box-refinement algorithm to converge to the local structure most similar to the reference structure.34,35 X-ray holography32,36 was used to prove the correctness of the experimental relative electron density profiles for the (34) Stroud, R. M.; Agard, D. A. Biophys. J. 1979, 25, 495. (35) Makowski, L. J. Appl. Crystallogr. 1981, 14, 160. (36) Smith, H. M. Principles of Holography; Wiley-Interscience: New York, 1969.

Vectorially-Oriented Cytochrome Oxidase Monolayers

Figure 4. (a) Experimental Patterson function, PSA, for selfassembled cytochrome oxidase. The origin has been offset horizontally to correspond with the first Ge peak in ∆Fexp for ease of comparison between the two. (b) Experimental relative electron density profile, ∆Fexp, for self-assembled cytochrome oxidase. (c) Refined model relative electron density profile, ∆Fmod, for self-assembled cytochrome oxidase. (d) Refined model absolute electron density, Fmod, for self-assembled cytochrome oxidase in units of electrons/Å3. See text for further details.

composite systems derived via X-ray interferometry. If the Ge layers within the reference profile structure of the Ge/Si multilayer substrates are sufficiently narrow (as is possible with MBE fabrication) and Aku is sufficiently large, then the unknown profile structure for the SAM or SAM/ protein overlayer is reconstructed with minimal distortion at the edge of the Patterson function, P(z), which is uniquely obtained by Fourier transformation of the kinematical meridional X-ray diffraction data without any phase information. Figure 4 summarizes the various stages in the determination of the correct profile structure of self-assembled cytochrome oxidase tethered to a multilayer substrate. Figure 4b shows the experimental relative electron density profile structure, ∆Fexp(z), for the composite Ge/Si multilayer substrate/amine SAM/self-assembled cytochrome oxidase system, derived by applying the constrained realspace refinement to the specimen’s meridional X-ray diffraction data using the reference structure as the primary constraint. The two sharpest peaks of density correspond to the Ge2 layers in the multilayer substrate. After the formation of the protected amine SAM, an additional feature was observable on the surface of the substrate. As previously described in detail by Prokop et al.,30 the protected amine SAM has a profile length of ∼17 Å, which is consistent with its structure. While the meridional X-ray diffraction data was not collected for the deprotected SAM due to its instability, its profile length would necessarily be less than 17 Å after the cleaving of the bulky tert-butoxycarbonyl groups. Indeed, the deprotected amine-terminated SAM can still clearly be seen in the absolute electron density profiles shown by Prokop et al., even after addition of the Ca2+-ATPase protein; the profile lengths of the amine-terminated SAM were found to range from 10-15 Å with respective electron densities

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of 0.13-0.2 electron/Å3, significantly less than that of ∼0.35 electron/Å3 for a densely packed methyl-terminated SAM.30 In the current system under investigation, after the formation of the self-assembled cytochrome oxidase layer on the surface of the SAM, additional, more complex features appear for 0 e z e 100 Å. To understand the nature and source of these new features, a real-space model on an absolute electron density scale was constructed to account for each feature. Figure 4d shows the refined model absolute electron density profile Fmod(z) for the Ge/ Si multilayer substrate/amine SAM/self-assembled cytochrome oxidase system, which accounts for each feature in ∆Fexp(z) (Figure 4b). This fact is demonstrated by a comparison of Figure 4b with Figure 4c, the model relative electron density profile, ∆Fmod(z), where ∆Fmod(z) is uniquely calculated by a “double” Fourier transform (i.e., Fourier transform-inverse Fourier transform) of Fmod(z) subject to the experimental qz window. A one-to-one correspondence between each feature in the refined model relative electron density profile and its counterpart in the experimental relative electron density profile established both the position ((0.1 Å) and electron density level ((0.01 electron/Å3) of each feature in the so-refined Fmod(z).31 The previously mentioned peak features corresponding to the Ge layers in the substrate are positioned at z = -85 and -40 Å in Figure 4d. The presence of a smaller peak at z = -25 Å in the electron density for the protected amine SAM system (not shown here) and in the refined absolute electron density model amine SAM/self-assembled cytochrome oxidase system (Figure 4d) which have an electron density greater than silicon suggests that some evolution of the substrate structure occurred during the alkylation process. Previous studies using 3(Ge2Si30) MBE substrates have reported multiple peaks in the region of the outer Ge2 layer which indicated migration of Ge toward the surface of the substrate.31 Regions between the Ge peaks having an electron density of 0.7 electron/Å3 represent the silicon layers. The broad Si/SiOx interface occurs at z = -12 Å. This SiOx layer is formed during the cleaning procedures prior to chemisorption of the amine SAM to the surface of the substrate.21 The SiOx/amine SAM interface is discernible at the profile position z = 0 Å in Figure 4d, giving rise to the slight shoulder feature at z = 4 Å in Figure 4c, while the SAM feature itself occurs within 0 < z < 10 Å of Figure 4d. This length is consistent with what we would expect (see ref 30) for the deprotected SAM, having a maximum possible length of ∼15 Å, giving rise to a smooth surface, at least relative to the much larger size of the cytochrome oxidase molecules. Hence, any electron density beyond this surface can be attributed to the addition of the protein, and in Figure 4d the cytochrome oxidase molecular profile can be seen to occur within 10 < z < 90 Å, giving rise to two asymmetric peaks centered at z = 15 Å and z = 60 Å which were not previously present. The peak centered at z = 15 Å is significantly smaller than the peak occurring at z = 60 Å, indicating that the majority of the electron density occurs within a broad (∼45 Å fwhm) feature positioned about 50 Å away from the SAM’s surface. The criteria for X-ray holography32,36 are satisfied for the amine SAM/self-assembled cytochrome oxidase system, and therefore the experimental electron density profile can be proven to be correct. Figure 4a is the Patterson function, P(z)SA, for z g 0 for the amine SAM/ self-assembled cytochrome oxidase system (the origin of which has been offset horizontally to correspond with the first Ge2 peak in ∆Fexp(z) for ease of comparison). Comparison of the features in Figure 4a,b over the region 0 e z e 110 Å reveals that the Ge2 peak feature at the left

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Figure 5. Meridional x-ray diffraction data, log[I(qz)], for (a) a typical bare two unit cell Ge/Si multilayer substrate and (b) for the same substrate plus amine-terminated SAM with LBdeposited cytochrome oxidase. The diffraction data at higher qz is also shown on an expanded scale, and dashed lines have been added to help guide the eye for comparative purposes. The abscissa is the reciprocal space coordinate, qz (Å -1), and the ordinate is the logarithm of counts collected.

edge of the relative electron density profile is convoluted with the amine SAM/self-assembled cytochrome oxidase overlayer features at the right edge of the profile to reconstruct these same features in the Patterson function over the same region of z. Therefore, the nearly identical agreement between the amine SAM/self-assembled cytochrome oxidase overlayer profile features at the edge of the Patterson function and those at the edge of the relative electron density profile indicates that the organic overlayer profile structure derived via X-ray interferometry is proven correct by X-ray holography. X-ray Interferometry/Holography: LangmuirBlodgett Case. The meridional X-ray diffraction data for another bare Ge/Si multilayer substrate and for this same substrate/amine-terminated SAM with LB-deposited cytochrome oxidase are shown in Figure 5 as log [I(qz)]. The data at higher qz have again been expanded 10-fold to display the scattering more clearly. Evidence of interference upon addition of the LB-deposited protein overlayer structure to the Ge/Si multilayer substrate is not so readily apparent in Figure 5 as it was for the selfassembled case in Figure 2, so for comparison, the data are also shown as their corrected meridional intensity functions in Figure 6. As for the self-assembled case, the term |Fkin(qz)|2 for the bare Ge/Si multilayer substrate, the substrate plus protected amine SAM, and the composite multilayer substrate/amine SAM/protein systems was obtained from the Lorentz-corrected, meridional elastic X-ray scattering, Ic(qz), by subtraction of |Fspec(qz)|2, as approximated by the Lorentz-corrected meridional scattering from a uniform silicon substrate21,31 and a Lorentz factor of qz was applied to correct for the ω-oscillations of the specimens.33 This procedure resulted in the kinematical diffraction data being restricted to the qz range (qz)min ≈ 0.008 e qz e (qz)max ≈ 0.069 Å-1 for the LB-deposited specimens. A Fourier transformation, restricted to this qz window, of the kinematical meridional X-ray diffraction data without any phase information yielded the Patterson function, P(z)LB, for the LB-deposited sample. Since the criteria for X-ray holography are also satisfied for the amine SAM/LB-deposited cytochrome

Edwards et al.

Figure 6. Corrected (see Results) meridional intensity functions, Ic(qz), for the data shown in Figure 5 for (a) a typical bare two unit cell Ge/Si multilayer substrate and (b) for the same substrate plus amine-terminated SAM with LB-deposited cytochrome oxidase. Dashed lines have been added to help guide the eye for comparative purposes.

Figure 7. (a) Experimental Patterson function for LB-deposited cytochrome oxidase, PLB. (b) Experimental Patterson function for self-assembled cytochrome oxidase, PSA.

oxidase system, we can directly compare the Patterson functions of the LB-deposited (Figure 7a) and selfassembled (Figure 7b) specimens. Obviously, while slightly different substrates were used for the two specimens resulting in some minor differences between the two Patterson functions for the shorter correlation lengths, e.g., 0 < z < 100 Å, there are strong similarities between the profile structures for the two cytochrome oxidase overlayers reconstructed near the edges of the two Patterson functions, e.g., 100 < z < 200 Å. In particular, the same profile features of the self-assembled cytochrome oxidase monolayer are broadened and diminished in amplitude for the LB-deposited cytochrome oxidase monolayer occurring over an increased profile length of ∼100 Å, as compared with ∼80 Å for the selfassembled case. Hence, the cytochrome oxidase molecules in these LB-deposited monolayers appear to possess the same vectorial orientation as the self-assembled monolayers. It is likely that the cytochrome oxidase molecules are already vectorially-oriented in the Langmuir monolayer with their large hydrophilic “cytoplasmic” domain oriented toward the subphase. This was anticipated, and hence the amine-terminated SAM substrates were withdrawn upward through the monolayer to tether the

Vectorially-Oriented Cytochrome Oxidase Monolayers

Figure 8. Polarized optical absorption spectra of a reduced, LB-deposited cytochrome oxidase monolayer at a 45° angle of incidence. The horizontally polarized Soret absorption peak being larger than the vertically polarized peak in this configuration indicates that the heme planes (and, therefore, the long axis of the average protein molecule) are more perpendicular than parallel to the substrate plane. From the figure, we can calculate a dichroic ratio (defined as the horizontal absorption divided by the vertical absorption) of approximately 1.2. At this angle of incidence, this corresponds to a tilt angle of about 65° between the average heme plane and the substrate plane.

cytochrome c binding region within the cytoplasmic domain to the amine-terminated SAM and provide a direct comparison with the self-assembled cytochrome oxidase specimens. Optical Linear Dichroism. An example of the optical linear dichroism observed for an LB-deposited specimen is shown in Figure 8. The polarized optical absorption spectra were taken with the protein specimen in a reduced state and at an angle of incidence of 45°. Here, the angle of incidence is defined as the angle of rotation between the incident beam and the normal to the specimen plane. The horizontal and vertical polarizations (defined in the laboratory frame) were, consequently, perpendicular and parallel to the axis of specimen rotation, respectively. The horizontally polarized Soret absorption peak being larger than the vertically polarized peak in this configuration confirms that the heme planes (and, therefore, the long axis of the average protein molecule which is known to be essentially parallel to both heme planes) are more perpendicular than parallel to the substrate plane. Spectra were also taken with an angle of incidence of 0° and found to be identical for both polarizations, as would be expected for a chromophore that is isotropically oriented about the normal to the substrate plane. From Figure 8, we can calculate a dichroic ratio (defined as the horizontal absorption divided by the vertical absorption) of approximately 1.2 for the 45° angle of incidence. This corresponds to a tilt angle of around 65° between the average heme plane and the substrate plane.29 No discernible dichroism, i.e., a dichroic ratio of 1.0 given the signal-to-noise level, was detected for any of the selfassembled specimens (see Discussion). Discussion The amine-terminated SAM surface was chosen to mimic the ring of lysine residues around the heme edge of cytochrome c which is known to be involved in the binding of this protein to the high-affinity site of cytochrome oxidase.37,38 The putative location of this binding (37) Capaldi, R. A.; Darley-Usmar, V.; Fuller, S.; Millett, F. FEBS Lett. 1982, 138, 1. (38) Capaldi, R. A. Annu. Rev. Biochem. 1990, 59, 569.

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site is toward the end of the cytochrome oxidase molecule which protrudes on the C (cytoplasmic) side of the membrane.28,37 Previous work, involving electron microscopic and X-ray diffraction analyses of two-dimensionally crystalline forms of the oxidase, has indicated that this side of the enzyme protrudes further from the membrane than the other [M (matrix)] side of the molecule does,39,40 while more recent results, involving X-ray crystallography of a three-dimensionally crystalline form, estimate the extramembrane domains protruding from the M and C sides of the membrane to be about equal in extension from the bilayer surface.41 Electrostatic interactions between this positively charged portion of cytochrome c and a negatively charged area of cytochrome oxidase are thought to be important in orienting the smaller protein as it approaches the C side of the oxidase molecule. The binding studies outlined earlier and characterized by optical spectroscopy demonstrate the reproducibility and reversibility of the protein-SAM interaction as well as its electrostatic nature. In addition, the absorbances are on the order of those expected for a single monolayer of cytochrome oxidase, the in-plane packing density being somewhat higher for the LB-deposited specimens as compared to that of their self-assembled counterparts. The formation of self-assembled cytochrome oxidase films on MBE substrates allowed the determination of the profile structure of the system by X-ray interferometry, and proof of the correctness of these so-derived profile structures by X-ray holography, to a spatial resolution of ∼10 Å. The profile lengths of the cytochrome oxidase feature, obtained from the absolute electron density model for the self-assembled specimens and estimated from the Patterson function in the LB-deposited case, in conjunction with the optical absorption spectroscopy results confirm that the cytochrome oxidase forms a single monolayer in both cases. The profile lengths of around 85 Å for the self-assembled monolayers and approximately 100 Å for the LB-deposited monolayers agree with those of Valpuesta13 (110 Å) and Tsukihara41 (117 Å) when tilting of the molecular long axis relative to the substrate normal is taken into account. The strong asymmetry of the cytochrome oxidase profile structure evidenced in the refined absolute electron density model (Figure 4d) is indicative of a high degree of vectorial orientation of the molecules within the monolayer that is much closer to unidirectional than bidirectional, although this asymmetry is not as pronounced as for the photosynthetic reaction center, Rhodopseudomonas sphaeroides,3 which was shown to be essentially unidirectional. The asymmetry can be seen more clearly in Figure 9a, which shows the experimental absolute electron density profile, obtained by summation of the mean electron density profile for the refined model absolute electron density profile, Fhmod(z), and the experimental relative electron density profile, ∆Fexp(z)ssee eq 1. This procedure eliminates any (qz)min truncation effects from the experimental electron density profile, leaving only the high spatial frequency components, due to the (qz)max truncation, in the profile. Figure 9b shows the corresponding refined model absolute electron density profile for comparison and indicates that the cytochrome oxidase profile might have been better modeled to extend slightly further from the amine-terminated SAM surface by ∼10 Å. (39) Deatherage, J. F.; Henderson, R.; Capaldi, R. A. J. Mol. Biol. 1982, 158, 487. (40) Jayaraman, U.; Chang, T.; Frey, T. G.; Blasie, J. K. Biophys. J. 1987, 51, 475. (41) Tsukihara, T.; Aoyama, H.; Yamashita, E.; Tomizaki, T.; Yamaguchi, H.; Shizawa-Itoh, K.; Nakashima, R.; Yaono, R.; Yoshikawa, S. Science 1995, 269, 1069.

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Figure 9. (a) Experimental absolute electron density profile for the self-assembled cytochrome oxidase specimen, produced by summation of the mean electron density profile, Fhmax (z), with the experimental relative electron density profile, ∆Fexp (z), to contain only (qz)max truncation effects. (b) Refined model absolute electron density, Fmod (z), for the self-assembled cytochrome oxidase specimen.

In order to further investigate the degree of vectorial orientation within these cytochrome oxidase single monolayers, the experimental absolute electron density profile for the self-assembled cytochrome oxidase monolayer was compared with the low-resolution three-dimensional structure for the oxidase molecule obtained from the electron microscopy of a two-dimensionally crystalline form of the oxidase,13 a form more similar to these single monolayers than the three-dimensionally crystalline form. Data from a two-dimensional crystal of frozen hydrated beef heart mitochondrial cytochrome oxidase was obtained from Dr. T. G. Frey13 and converted to a format suitable for manipulation (CCP4 format42). A box-shaped mask, just slightly larger than one unidirectional dimer from the electron density map contoured at 1σ (where σ is the rms deviation from the mean electron density), was constructed using the program MAMA43 so that all points of non-zero electron density were included in the volume of the mask. The program O44 was used to edit this mask so that it excluded any electron density that appeared to be from other nearby dimers. Gaps between the positive parts of the mask were then filled in using the program MAMA to result in a single solid mask, the volume of which completely contained a single dimer contoured at this level. This mask was then applied to the full CCP4 format electron density map using the program AVE,43 effectively isolating one dimer from the map. It was then possible to use the program MAPMAN43 to produce a onedimensional projection of the masked volume onto the z-axis as shown in Figure 10a. The entire masked electron density, i.e., irrespective of contouring level, was used in this integration over the planes perpendicular to the z-axis so that the resulting plot is analogous to what we see from X-ray interferometry, only for a unidirectional dimer of (42) Collaborative Computing Project, Number 4 (CCP4), U.K. Biotechnology and Biological Sciences Research Council, Acta Crystallogr. D. 1994, 50, 760. (43) MAMA: Kleywegt, G. J., Jones, T. A., Department of Molecular Biology, University of Uppsala, Sweden. (44) O: Jones, T. A.; Kjeldgaard, M., Department of Molecular Biology, University of Uppsala, Sweden and Department of Chemistry, Aarhus University, Denmark. Jones, T. A.; Zou, J. Y.; Cowan, S. W.; Kjeldgaard, M. Acta.Crystallogr. A 1991, 47, 110.

Figure 10. (a) Projection onto the z-axis of the integration (over the planes perpendicular to the z-axis) of the electron density of one unidirectional dimer of cytochrome oxidase as obtained from the three-dimensional electron microscopy data. See text for further details. (b) Expanded protein overlayer portion of the experimental absolute electron density profile for the self-assembled cytochrome oxidase monolayer specimen.

cytochrome oxidase rather than a monolayer. The protein overlayer portion of the experimental absolute electron density profile for the self-assembled cytochrome oxidase monolayer is shown for comparison in Figure 10b and is strikingly similar to the projected density obtained from the electron microscopy data. The two z-axis scales are not the same, but rather the experimental absolute electron density profile is shown on such a scale as to give the best visual comparison of the two plots. This corresponds to an average cytochrome oxidase profile length of around 80 Å as reported earlier for the self-assembled specimens. This shorter profile length, as compared to the 110 Å result reported by Valpuesta et al.,13 is indicative of some degree of tilting of the long axis of the protein with respect to the substrate plane. However, this profile length is an average, and thus the profile features of the oxidase molecule shown in Figure 10b would be expected to be broadened relative to those of Figure 10a. Nevertheless, the overall close similarity of the two profiles in Figure 10, including the significantly higher electron density toward the end of the profile furthest from the amine-terminated SAM surface, confirms a vectorial orientation approaching unidirectional within these tethered single monolayers. Since the tethered monolayer is expected to be more like the two-dimensionally crystalline form than the three-dimensionally crystalline form, the location of the region of higher electron density also implies that it is the C end of the molecule that is binding to the amine-terminated SAM, as would be expected. While optical linear dichroism cannot assess the vectorial orientation of the protein molecules within such

Vectorially-Oriented Cytochrome Oxidase Monolayers

tethered single monolayers, our results are fully consistent with the orientation of the long axis of the cytochrome oxidase molecule relative to the monolayer plane determined by the X-ray interferometry/holography. The optical linear dichroism results for the LB-deposited specimens indicate that the heme planes (and, therefore, the long axis of the average protein molecule) are more perpendicular than parallel to the substrate plane with an average tilt angle of 65° (between the average heme plane and substrate plane). If the long axis of a cytochrome oxidase molecule is actually 110-117 Å (on the basis of the electron microscopy13 and X-ray crystallography41 results), this tilt would result in a profile length of 100106 Å for the LB-deposited cytochrome oxidase monolayer which agrees well with that estimated from the Patterson function for the LB specimen. Also, a dichroic ratio of 1.2 in the Soret band is comparable to the 1.11-1.13 minimum dichroic ratios reported in a previous study29 for the Soret band of thick oriented multilayers of membranous cytochrome oxidase at a 45° angle of incidence. In this previous report, dichroic ratios from the 598 nm band (too weak to be seen in the current, single monolayer study) appeared significantly larger (1.37-1.61) than those observed in the Soret. No measurable dichroism, i.e., a dichroic ratio of 1.0 within the signal-to-noise level, was detected for any of the self-assembled specimens. However, the measured profile length of about 85 Å for the selfassembled cytochrome oxidase monolayer, as determined from its unique electron density profile, would indicate that the long axis of the oxidase molecule and, hence, the average heme plane would be tilted approximately 51° relative to the substrate plane, close to the so-called “magic angle” of 54.7° which results in a dichroic ratio of 1.0 at a 45° angle of incidence. Extensive investigations of enzyme functionality were outside the scope of this present study. Nevertheless, redox potential measurements are currently in progress, and initial results on LB-deposited cytochrome oxidase monolayer specimens indicate that the oxidase heme a and a3 midpoint potentials are similar to those for the native enzyme.45 (45) Haas, A.; Dutton, P. L. work in progress.

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Conclusion We have demonstrated that detergent-solubilized mitochondrial cytochrome c oxidase can be electrostatically tethered to amine-terminated SAM surfaces via selfassembly from solution. Comparison of the electron density profiles of the so-tethered oxidase monolayers along the axis normal to the substrate plane with the analogous profiles derived from the 3D structure as obtained via electron microscopy of a 2D crystalline form of the oxidase has indicated that it is the cytoplasmic C end of the molecule that binds to the amine-terminated SAM, thereby producing a unidirectional vectorial orientation of the enzyme molecules in the monolayer. Furthermore, the electron density profiles for monolayers of detergent-solubilized cytochrome oxidase LB-deposited on amine-terminated SAMs have indicated a vectorial orientation for the oxidase molecules similar to the selfassembled case. In addition, the LB monolayers tend to be more densely packed than their self-assembled counterparts, with the normal to the average heme plane lying more parallel to the substrate than for monolayers formed by self-assembly. These structurally-characterized, vectorially-oriented single monolayers of cytochrome oxidase are now wellsuited to directly correlated structure-function studies concerning transmonolayer electrochemical phenomena. Such structural studies via X-ray interferometry/holography have also recently been extended to the cytochrome c/cytochrome oxidase bimolecular complex.46 Acknowledgment. This work was supported by National Institutes of Health Grant GM33525 to J.K.B. A.M.E. would especially like to thank Charles Lesburg and Gerard Kleywegt for their assistance with the crystallography programs. Supporting Information Available: Details of the cytochrome oxidase isolation and purification procedure (3 pages). Ordering information is given on any current masthead page. LA960769I (46) Edwards, A. M.; Blasie, J. K.; Bean, J. C. submitted to Biophys. J., October 1996.