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• W2074564151 abstract "The electron transfer complex between bovine cytochrome c oxidase and horse cytochrome c has been predicted with the docking program DOT, which performs a complete, systematic search over all six rotational and translational degrees of freedom. Energies for over 36 billion configurations were calculated, providing a free-energy landscape showing guidance of positively charged cytochrome c to the negative region on the cytochrome c oxidase surface formed by subunit II. In a representative configuration, the solvent-exposed cytochromec heme edge is within 4 Å of the indole ring of subunit II residue Trp104, indicating a likely electron transfer path. These two groups are surrounded by a small, hydrophobic contact region, which is surrounded by electrostatically complementary hydrophilic interactions. Cytochrome c/cytochrome c oxidase interactions of Lys13 with Asp119 and Lys72 with Gln103 and Asp158 are the most critical polar interactions due to their proximity to the hydrophobic region and exclusion from bulk solvent. The predicted complex matches previous mutagenesis, binding, and time-resolved kinetics studies that implicate Trp104 in electron transfer and show the importance of specific charged residues to protein affinity. Electrostatic forces not only enhance long range protein/protein association; they also predominate in short range alignment, creating the transient interaction needed for rapid turnover. The electron transfer complex between bovine cytochrome c oxidase and horse cytochrome c has been predicted with the docking program DOT, which performs a complete, systematic search over all six rotational and translational degrees of freedom. Energies for over 36 billion configurations were calculated, providing a free-energy landscape showing guidance of positively charged cytochrome c to the negative region on the cytochrome c oxidase surface formed by subunit II. In a representative configuration, the solvent-exposed cytochromec heme edge is within 4 Å of the indole ring of subunit II residue Trp104, indicating a likely electron transfer path. These two groups are surrounded by a small, hydrophobic contact region, which is surrounded by electrostatically complementary hydrophilic interactions. Cytochrome c/cytochrome c oxidase interactions of Lys13 with Asp119 and Lys72 with Gln103 and Asp158 are the most critical polar interactions due to their proximity to the hydrophobic region and exclusion from bulk solvent. The predicted complex matches previous mutagenesis, binding, and time-resolved kinetics studies that implicate Trp104 in electron transfer and show the importance of specific charged residues to protein affinity. Electrostatic forces not only enhance long range protein/protein association; they also predominate in short range alignment, creating the transient interaction needed for rapid turnover. cytochromec oxidase(s) cytochrome c root mean square cytochrome c peroxidase The determination of structures of bacterial and mitochondrial cytochrome c oxidases (CcO)1 by x-ray crystallography (1Iwata S. Ostermeier C. Ludwig B. Michel H. Nature. 1995; 376: 660-669Crossref PubMed Scopus (1985) Google Scholar, 2Ostermeier C. Harrenga A. Ermler U. Michel H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10547-10553Crossref PubMed Scopus (717) Google Scholar, 3Tsukihara T. Aoyama H. Yamashita E. Tomizaki T. Yamaguchi H. Shinzawa-Itoh K. Nakashima R. Yaono R. Yoshikawa S. Science. 1995; 269: 1069-1074Crossref PubMed Scopus (1295) Google Scholar, 4Tsukihara T. Aoyama H. Yamashita E. Tomizaki T. Yamaguchi H. Shinzawa-Itoh K. Nakashima R. Yaono R. Yoshikawa S. Science. 1996; 272: 1136-1144Crossref PubMed Scopus (1936) Google Scholar, 5Yoshikawa S. Shinzawa-Itoh K. Nakashima R. Yaono R. Yamashita E. Inoue N. Yao M. Fei M.J. Libeu C.P. Mizushima T. Yamaguchi H. Tomizaki T. Tsukihara T. Science. 1998; 280: 1723-1729Crossref PubMed Scopus (976) Google Scholar) has opened the door to understanding the structural basis for its function in respiratory chain activity. One step of the respiratory chain involves the transfer of electrons from cytochrome bc 1 complex to CcO, both membrane-bound complexes, via the water-soluble protein cytochromec (Cc). Efficient electron transfer requires both rapid complex formation and rapid product dissociation. Determination of the structure of the complex formed between CcO and Cc would reveal the interactions promoting this transient interaction as well as possible paths for intermolecular electron transfer. The complex structure, however, has yet to be determined, in part due to the difficulties in crystallizing membrane-bound proteins and possibly also due to the transient nature of the interaction. On the other hand, this redox system may be particularly amenable to computational search methods based on static crystallographic structures because of the fast kinetics of these interactions, which suggest that large conformational changes do not occur upon complex formation. In addition, computational techniques may shed light on long range electrostatic guidance between the two partners and on the dynamic nature of the interaction. The docking program DOT (6Ten Eyck L.F. Mandell J. Roberts V.A. Pique M.E. Hayes A. Simmons M. Proceedings of the 1995 ACM/IEEE Supercomputing Conference, San Diego, December 3–8, 1995. IEEE Computer Society Press, Los Alamitos, CA1995http://www.supercomp.org/sc95/proceedings/636_LTEN/SC95.HTMGoogle Scholar) provides a complete search of all orientations between two rigid molecules by systematically rotating and translating one molecule about the other, a procedure considered computationally unfeasible just a few years ago (7Kuntz I.D. Science. 1992; 257: 1078-1082Crossref PubMed Scopus (899) Google Scholar). At that time, the extent of the search was limited to, at most, energy calculations for a few million configurations, as in the programs TURNIP (8Roberts V.A. Freeman H.C. Olson A.J. Tainer J.A. Getzoff E.D. J. Biol. Chem. 1991; 266: 13431-13441Abstract Full Text PDF PubMed Google Scholar) and DOCK (9Shoichet B.K. Kuntz I.D. Protein Eng. 1993; 6: 723-732Crossref PubMed Scopus (187) Google Scholar). Alternately, the representation of the moving molecule was greatly simplified to a few point charges (10Northrup S.H. Boles J.O. Reynolds J.C.L. Science. 1988; 241: 67-70Crossref PubMed Scopus (270) Google Scholar, 11Warwicker J. J. Mol. Biol. 1989; 206: 381-395Crossref PubMed Scopus (37) Google Scholar). DOT systematically samples configurations for two rigid molecules over all space and expresses the interaction energies as correlation functions, which are rapidly computed using fast Fourier transforms. The electrostatic potentials used by DOT are calculated by Poisson-Boltzmann methods (12Gilson M.K. Honig B. Proteins. 1988; 3: 32-52Crossref PubMed Scopus (267) Google Scholar, 13Gilson M.K. Davis M.E. Luty B.A. McCammon J.A. J. Phys. Chem. 1993; 97: 3591-3600Crossref Scopus (299) Google Scholar, 14Bashford D. Ishikawa Y. Oldehoeft R.R. Raynders J.V. Tholburn M. Scientific Computing in Object-Oriented Parallel Environments. Springer, New York1997: 233-240Google Scholar), which take both solvent and ionic strength effects into account. Because DOT performs a complete search, both long range and short range interactions can be examined. Here, we describe the application of DOT to the interaction of bovine CcO with horse Cc. The calculated complex identifies the CcO Trp104(Trp143) 2All residue numbering in CcO is according to the bovine sequence with R. sphaeroides residues given in parentheses. side chain as the point of electron entry into CcO and implicates specific charged side chains in protein-protein association, in agreement with mutagenesis, binding, and time-resolved kinetics studies (15Witt H. Malatesta F. Nicoletti F. Brunori M. Ludwig B. Eur. J. Biochem. 1998; 251: 367-373Crossref PubMed Scopus (80) Google Scholar, 16Wang K. Zhen Y. Sadoski R. Grinnell S. Geren L. Ferguson-Miller S. Durham B. Millett F. J. Biol. Chem. 1999; 274: 38042-38050Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 17Zhen Y. Hoganson C.W. Babcock G.T. Ferguson-Miller S. J. Biol. Chem. 1999; 274: 38032-38041Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Properties of the calculated interface resemble those found in the crystallographic structure of the electron transfer complex of yeast cytochrome c peroxidase (CcP) with yeast Cc (18Pelletier H. Kraut J. Science. 1992; 258: 1748-1755Crossref PubMed Scopus (712) Google Scholar), suggesting a general structural motif for transient protein-protein interactions. Coordinates for bovine CcO (1OCC) and horse Cc (1HRC) were obtained from the Protein Data Bank (19Bernstein F.C. Koetzle T.F. Williams G.J.B. Meyer Jr., E.F. Brice M.D. Rodgers J.R. Kennard O. Shimanouchi T. Tasumi M. J. Mol. Biol. 1977; 112: 535-542Crossref PubMed Scopus (8182) Google Scholar).Rhodobacter sphaeroides cytochrome c 2was also investigated because its interactions with CcO have been examined (16Wang K. Zhen Y. Sadoski R. Grinnell S. Geren L. Ferguson-Miller S. Durham B. Millett F. J. Biol. Chem. 1999; 274: 38042-38050Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 17Zhen Y. Hoganson C.W. Babcock G.T. Ferguson-Miller S. J. Biol. Chem. 1999; 274: 38032-38041Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Polar hydrogen atoms were added with the computer graphics program Insight (MSI Inc., San Diego). Partial atomic charges for protein atoms and heme groups were based on the AMBER library that includes polar hydrogen atoms only (20Weiner S.J. Kollman P.A. Case D.A. Singh U.C. Ghio C. Alagona G. Profeta Jr., S. Weiner P. J. Am. Chem. Soc. 1984; 106: 765-784Crossref Scopus (4895) Google Scholar). Histidine residues were neutral with a single proton on N-ε unless metal ligation or salt-bridge formation indicated otherwise. For horse Cc, the C terminus was negatively charged, the N terminus was neutral because of acetylation, and the heme group had an overall charge of −2, resulting in a total charge of +6. Of the three available coordinate sets forR. sphaeroides cytochrome c 2 (1CXA, 2CXB, and 1CXC), 1CXC was used because it is the most complete and determined at the highest resolution (1.6 Å). Missing atoms for residues 10, 32, 33, 35, 74, 78, 85, 89, 95, 97, 105, and 106 were added with Insight while using other two structures as guides. Both termini were charged, and both His75 and His111were doubly protonated to satisfy intramolecular salt bridges, giving a total charge of 0. An overall charge of −2 was previously used (21Tiede D.M. Vashishta A.-C. Gunner M.R. Biochemistry. 1993; 32: 4515-4531Crossref PubMed Scopus (114) Google Scholar), possibly because all His residues were left neutral. CcO presents a challenging computational problem because of its large size (∼204,000 kDa). Since Cc interacts with CcO in the mitochondrial intermembrane space, only this region of CcO was used in the calculation. The bovine CcO complex was cut by a plane placed below the ring of positively charged residues that presumably lie at the inner mitochondrial membrane surface (See Fig. 1 C). All residues with any atom above this plane were retained. The selected coordinates included all of subunit II, except the transmembrane helical segment, and parts of subunits I, IV, VIa, VIb, VIc, VIIb, VIIc, and VIII. The heme groups and the other bound metal ions lie in the membrane-bound portion of the molecule and were not included. Polypeptide termini resulting from cutting out the membrane-bound region were kept neutral, while standard N termini and C termini were charged. Each copper atom of the dicopper site CuA in subunit II was assigned a charge of +1.5, giving the metal cluster a total charge of +3 (22Beinert H. Eur. J. Biochem. 1997; 245: 521-532Crossref PubMed Scopus (115) Google Scholar). The total charge on the selected bovine CcO coordinates was −7. Electrostatic potentials were calculated with the program UHBD (13Gilson M.K. Davis M.E. Luty B.A. McCammon J.A. J. Phys. Chem. 1993; 97: 3591-3600Crossref Scopus (299) Google Scholar), which uses finite difference methods to solve the Poisson-Boltzmann equations for electrostatic potential. Potentials were calculated on a grid of the same size and spacing used in the DOT calculation: 128 Å on a side with 1-Å spacing. A dielectric of 3 for the protein, a dielectric of 80 for the surrounding environment, an ion exclusion radius of 1.4 Å, and an ionic strength of 50 mm were used. In the DOT calculation, Cc was assigned as the moving molecule and CcO as the stationary molecule. The shape distribution of Cc was represented by a value of 1 at each atomic position. Note that with this description, the volume of the moving molecule is represented by its atomic coordinates rather than by its full van der Waals volume. The charge distribution of Cc was represented by partial charges at the atomic positions of all nonhydrogen and polar hydrogen atoms, with charges taken from the AMBER library. The selected coordinates of CcO were centered in the x andy directions on a cubical grid 128 Å on a side with 1-Å grid spacing (about 2.1 million total grid points). The coordinates were moved in the z direction to position the approximate plane of the membrane near the bottom of the box, increasing the region representing the surrounding intermembrane space that Cc can occupy. The shape of CcO was represented by an excluded van der Waals volume surrounded by a 3.0 Å layer of favorable potential. To map the shape potential of CcO onto the grid, grid points within 4.5 Å of all nonhydrogen atoms were assigned a value of −1, and then grid points within 1.5 Å of all nonhydrogen atoms were assigned a highly unfavorable value of 1000. All other grid points were assigned a value of 0. Since the electrostatic potential is calculated only once for the stationary molecule, sophisticated, computationally expensive methods can be used. The Poisson-Boltzmann electrostatic potential for CcO was calculated with UHBD. It was assumed that an aqueous environment surrounded the selected CcO coordinates; the low dielectric of the membrane was not taken into account. Since the stationary molecule is represented by its van der Waals volume but the moving molecule is represented by its atomic centers, some atoms of the moving molecule can approach within 1.5 Å of the stationary molecule atom centers. This accommodates small conformational changes induced upon complex formation. Unfortunately, too close an approach can also result in an artificially large electrostatic contribution when a moving molecule atom lies between the molecular and the solvent-accessible surfaces of the stationary molecule. Large changes in magnitude and distribution of electrostatic potential occur in the region between the two surfaces. The most intense potential on the molecular surface is concentrated on protuberances (termini of Lys, Arg, Asp, and Glu side chains), whereas the potential on the solvent-accessible surface is more delocalized, with the most intense potential generally concentrated in concave regions of the protein (see Fig. 1, A, B, andD). To alleviate this problem, the electrostatic potential grid values of CcO were clamped based on the extreme potentials found at the solvent-accessible surface, which represents the closest approach of the center of a water molecule (∼2.9 Å between atomic centers). For CcO, the upper limit was +4 kcal/mol/e and the lower limit was −6.0 kcal/mol/e. We initially used limits of ± 15 kcal/mol/e, the extremes found on protein molecular surfaces, but these limits gave a much larger number of favorable-energy false positives in DOT calculations. All energies computed by DOT are derived by placing the moving molecule in a potential field generated by the fixed molecule. The total energy for each configuration of the two molecules is the sum of the electrostatic and van der Waals energy terms, each of which can be described as a correlation function. Given a potential fieldS(r) describing the stationary molecule and a probe function M(r) describing the moving molecule, the energy is given by the following. E=∫M(r)S(r)dr(Eq. 1) If the moving molecule is rotated by an angle ϑ and displaced from the origin by a vector r 0, the energy of the system is given by the following. Eϑ(r0)=∫Mϑ(r−r0)S(r)dr(Eq. 2) Mathematically, this is the correlation of the potential fieldS(r) and the rotated functionM(r). For the electrostatic energy term,S(r) is the electrostatic potential field of CcO and M(r) is the set of partial charges for Cc. For the van der Waals term, S(r) is the shape potential for CcO described above. Each Cc atom within the favorable layer surrounding CcO contributes −0.1 kcal/mol to the interaction energy. A Cc atom lying within the van der Waals volume of CcO contributes a large unfavorable value to the interaction energy. To allow for side chain rearrangement induced upon complex formation, a user-specified number of moving molecule atoms can penetrate the volume of the stationary molecule without incurring an energy penalty, but no penetrations were allowed in these runs. This method for computing overlap is similar to that previously described (23Katchalski-Katzir E. Shariv I. Eisenstein M. Friesem A.A. Aflalo C. Vakser I.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2195-2199Crossref PubMed Scopus (866) Google Scholar, 24Vakser I.A. Aflalo C. Proteins. 1994; 20: 320-329Crossref PubMed Scopus (171) Google Scholar) except that here S(r) has been constructed to return a composite number containing both a count of collisions and a count of favorable interactions. Correlations can be calculated very efficiently through the use of the Convolution Theorem. The electrostatic and nonbonded energy functions are described on a grid of N points (1283). Evaluating the correlation directly requires N ×N multiplications, in our case 1286. Evaluating the correlation using fast Fourier transforms costs three fast Fourier transforms, each proportional to NlogN, andN multiplications, so the computational cost is proportional to N(3logN + 1), in our case 1283 × 20, or a savings of about 105. In the systematic search performed by DOT, Cc is centered at all grid points and then rotated, and the calculation is repeated. The full search performed here uses a grid of 1283 points and 17,354 distinct rotational orientations, resulting in ∼36 billion configurations between Cc and CcO. DOT maintains two 1283interaction grids. In the minimum-energy grid, the rotational orientation with the most favorable energy at each grid point is kept. The second grid, the free-energy grid, represents a free-energy landscape of intermolecular energies. To obtain the free-energy grid, the Boltzmann-weighted sum of the energies over all orientations at each grid point is calculated to give the partition sum as follows, Qj=∑i=1Re−Ei/kBT(Eq. 3) where j is a grid point, R is the number of angles through which the moving molecule is rotated, T is the temperature in degrees Kelvin, and k B is the Boltzmann constant. Q j is then converted to the Helmholtz free energy as follows. Aj=−kBTlogQj(Eq. 4) Electrostatic potentials for CcO and Cc were derived by integration of the Poisson-Boltzmann equation, which accurately takes into account the ionic strength of the surrounding medium and the dielectric boundaries between protein and solvent. Accurate representation of electrostatic forces is particularly important for electron transfer systems because of the role of electrostatics in both short range orientation and long range guidance, as suggested by the strong dependence of the reactions on ionic strength. Electrostatic potentials were calculated at a low ionic strength (50 mm), where Cc and CcO show strong binding (17Zhen Y. Hoganson C.W. Babcock G.T. Ferguson-Miller S. J. Biol. Chem. 1999; 274: 38032-38041Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). R. sphaeroides cytochromec 2 has a slower electron transfer rate withR. sphaeroides CcO than does horse Cc (17Zhen Y. Hoganson C.W. Babcock G.T. Ferguson-Miller S. J. Biol. Chem. 1999; 274: 38032-38041Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). To investigate the source of this difference, we calculated the electrostatic potential for both molecules, as has been done previously (21Tiede D.M. Vashishta A.-C. Gunner M.R. Biochemistry. 1993; 32: 4515-4531Crossref PubMed Scopus (114) Google Scholar). In horse Cc, the Lys residues surrounding the exposed heme edge create patches of strong positive potential (Fig. 1 A). The opposite face of Cc is generally neutral, creating a dipole (21Tiede D.M. Vashishta A.-C. Gunner M.R. Biochemistry. 1993; 32: 4515-4531Crossref PubMed Scopus (114) Google Scholar). The sequence of R. sphaeroides cytochrome c 2 is very different from that of horse Cc, including the loss of lysine at positions 13 and 72, resulting in a very different distribution of positive potential on the face of the molecule surrounding the exposed heme edge (Fig. 1 B). Since Cc and CcO interact in the intermembrane space, only that region of CcO along with the adjacent membrane-embedded portion (Fig. 1 C) was used (see “Experimental Procedures”). The selected coordinates include the subunit II dicopper CuA site, the initial site of electron transfer from Cc (25Hill B.C. J. Biol. Chem. 1991; 266: 2219-2226Abstract Full Text PDF PubMed Google Scholar, 26Geren L.M. Beasley J.R. Fine B.R. Saunders A.J. Hibdon S. Pielak G.J. Durham B. Millett F. J. Biol. Chem. 1995; 270: 2466-2472Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 27Malatesta F. Nicoletti F. Zickermann V. Ludwig B. Brunori M. FEBS Lett. 1998; 434: 322-324Crossref PubMed Scopus (24) Google Scholar). The most significant electrostatic feature is a large patch of negative potential formed by a concentration of Asp and Glu side chains in subunit II (Fig. 1 D). The greatest concentration of negative potential lies in a shallow pocket between Asp119 (Glu157) and Glu109 (Glu148). Within this negative patch is a hydrophobic surface loop that lies over the CuA site and consists of subunit II residues His102(Tyr141), Gly103 (Gly142), Trp104 (Trp143), and Tyr105(Tyr144). The rest of the CcO surface is generally neutral with small regions of positive and negative potential. DOT centers each rotational orientation of one molecule, the moving molecule, at each position on a grid that surrounds a second molecule, the stationary molecule. For each configuration of the two molecules, the intermolecular energy, a sum of electrostatic and van der Waals terms, is calculated (see “Experimental Procedures”). The time required for the calculation is independent of the number of atoms in either molecule and instead depends on the size of the grid and the number of rotational orientations of the moving molecule. The mathematics of the DOT calculation requires periodic boundary conditions; the grid is repeated in all directions over all space. Therefore, the grid must be large enough that the stationary molecule potentials are close to zero at the grid boundary. In addition, at least one diameter of the moving molecule about the stationary molecule must fit within the grid. We have found that a grid spacing of 1 Å provides a sufficiently accurate description of the stationary molecule and a reasonable fineness for the translation search. A cubical grid of 128 Å on each side fulfilled the size requirements for the interaction of the CcO fragment with Cc. The larger molecule, CcO, was assigned as the stationary molecule, and the smaller Cc was assigned as the moving molecule. This assignment provided two advantages. First, it allowed use of a finer spacing for the 128 × 128 × 128 grid, since the minimum dimension of the grid must be at least 2M + S, whereM and S represent the diameters of the moving and stationary molecules. Second, a smaller molecule will be sampled more finely over its surface for a given set of rotations. Our set of 17,354 rotational orientations provides ∼9° sampling, which corresponds to about a 2-Å spacing on the Cc surface. Storage requirements make it impractical to retain the energies and positions of all 36 billion configurations (1283 × 17,354) calculated. Instead, two types of interaction summary grids are maintained (see “Experimental Procedures”). The minimum-energy grid contains the energy, position, and orientation for the configuration (out of a possible 17,354) with the most favorable energy at each grid point. The free-energy grid provides a free-energy landscape combining information from all energy calculations. This grid displays a smoother change in energy across the grid than the minimum-energy grid, but rotational information is not retained. Both grids can be calculated simultaneously in about 7 h using 20 Sun Ultra-1 workstations running in parallel. A preliminary test with a single proton as the moving molecule demonstrated that the CcO shape and electrostatic potentials were properly aligned on the grid. A complete rotational search with 17,354 rotational orientations was then performed with DOT. Although our description of CcO allows Cc to move into the region of the grid that should be occupied by the membrane and membrane-bound portions of CcO, this area was electrostatically neutral, making favorable-energy interactions here unlikely. The top 2000 solutions of the minimum-energy grid (Fig. 2 A) form a single large group over the negative patch of CcO with a very few scattered outliers. The long axis of this group is approximately perpendicular to the membrane plane, with the most densely populated area nearest the membrane and CcO residue Trp104 (Trp143). The top 30 configurations of the minimum-energy grid show a more localized spatial distribution and had interaction energies ranging from −27.2 to −24.5 kcal/mol. Of these 30 solutions, 29 lie over the patch of negative potential on the CcO surface, with 25 having their exposed heme edge generally oriented toward the CcO molecule (Fig. 2 B). Within the top 30 configurations, there is a single, tight cluster consisting of five solutions with two nearby outliers (Fig. 2 B) having energies ranging from −26.5 to −24.9 kcal/mol. Among the closest five, rotations vary by 8–30°, and root mean square (r.m.s.) deviations for all nonhydrogen atoms range from 2.8 to 5.5 Å (average r.m.s. deviation = 4.1 Å). The two outliers have an r.m.s. deviation of less than 6 Å with at least one of the other five solutions. The r.m.s. deviations of the heme atoms are generally smaller, ranging from 1.8 to 5.5 Å (average r.m.s. deviation = 3.4 Å) for the closest five solutions, with the two outliers having an r.m.s. deviation of less than 4.5 Å with one of the five. The tighter clustering of the heme rings relative to the whole molecule indicates that the fit is best at the interface. All seven Cc solutions in this cluster have their exposed heme edge near the indole ring of CcO residue Trp104 (Trp143). In the free-energy grid, Cc solutions having the most favorable energies (Fig. 2 C) are concentrated over the negative patch of CcO, forming a two-lobed grouping. One lobe is centered about the best-energy cluster found in the top 30 configurations of the minimum-energy grid, and the other lobe is further from the membrane. This second lobe may indicate a weaker binding site for Cc. In both the free-energy and minimum-energy grids, favorable interactions extend from the large surface patch of negative potential on CcO out into solution, showing the effect of long range electrostatic guidance. The five configurations in the single, large cluster (Fig. 2 B) are energetically equivalent (within 1.0 kcal/mol in energy). From this group, the Cc configuration with the shortest distance from its exposed heme edge to CcO (ranked 19th in energy) was examined for specific intermolecular interactions. Distances are approximate, since rigid structures from individually determined structures were used and complex formation will cause some local rearrangement. The interface shows a central, relatively hydrophobic region surrounded by polar interactions. The exposed Cc heme edge is within 4 Å of the indole ring of CcO residue Trp104 (Trp143) (Table I, Fig. 3 A), supporting the role of Trp104 (Trp143) as the site of electron entry (15Witt H. Malatesta F. Nicoletti F. Brunori M. Ludwig B. Eur. J. Biochem. 1998; 251: 367-373Crossref PubMed Scopus (80) Google Scholar, 16Wang K. Zhen Y. Sadoski R. Grinnell S. Geren L. Ferguson-Miller S. Durham B. Millett F. J. Biol. Chem. 1999; 274: 38042-38050Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 17Zhen Y. Hoganson C.W. Babcock G.T. Ferguson-Miller S. J. Biol. Chem. 1999; 274: 38032-38041Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The hydrophobic region of Cc consists of the exposed heme edge, the adjacent Gln16 and Cys17 side chains, and residues 81–83, which form a β-strand paralleling one heme face (Fig. 1 A). This region contacts a corresponding hydrophobic region of CcO (Fig. 3, A and B) consisting of residues 102–105 (R. sphaeroides residues 141–144), which form a loop over the CuA site, the adjacent residues Tyr121 (Tyr159) and Asn203(Ser259), and backbone atoms of CcO Glu157(Ala213) and Asp158 (Asp214) (Table II).Table IIntermolecular distances in the CcO·Cc complexBovine CcOHorse Cc atomDistanceÅInteractions of the Cc heme and Cys17 ligandCuACu1Heme iron17.8CuA Cu2Heme iron18.9Trp104CE3Heme CBC3.8Trp104 CZ3Heme CBC3.3Trp104 CE3Cys17 SG6.0Trp104 CZ3Cys17 SG5.8Polar interactionsGln103 OE1aThis atom is assigned as NE2 in the crystallographic coordinates.Lys72NZ3.6 (11.9)bCA-CA distances are shown in parentheses.Glu109 OE1Lys86NZ2.4 (11.0)Glu109 OE1Lys87NZ3.1 (10.8)Asp119 OD2Lys13NZ2.3 (8.6)Asp139 OE1Gln12NE2" @default.
• W2074564151 created "2016-06-24" @default.
• W2074564151 creator A5075428131 @default.
• W2074564151 creator A5083070438 @default.
• W2074564151 date "1999-12-01" @default.
• W2074564151 modified "2023-09-29" @default.
• W2074564151 title "Definition of the Interaction Domain for Cytochrome con Cytochrome c Oxidase" @default.
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• W2074564151 doi "https://doi.org/10.1074/jbc.274.53.38051" @default.
• W2074564151 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10608874" @default.
• W2074564151 hasPublicationYear "1999" @default.
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