Eur. J. Biochem. 267, 755±766 (2000) q FEBS 2000
Solution structure of oxidized microsomal rabbit cytochrome b5 Factors determining the heterogeneous binding of the heme Lucia Banci, Ivano Bertini, Antonio Rosato and Serena Scacchieri
Cytochrome b5 is heterogeneous in solution because of the presence of two isomers (A and B), differing in the rotation of the heme plane around the axis defined by the a and g meso protons. For rabbit cytochrome b5, the A/B ratio is 5 : 1. The solution structure of the major form of the oxidized soluble fragment of rabbit microsomal cytochrome b5 (94 amino acids) is here solved through NMR spectroscopy. From 1908 NOEs, of which 1469 were meaningful, there were 246 pseudocontact shifts and 18 3J couplings, a family of 40 energyminimized conformers were obtained with average backbone rmsd (for residues 4±84) of 0.060 ^ 0.016 nm and average target function of 0.0078 nm2, no distance violations being larger than 0.03 nm. The structure was compared with the solution structures of the A (major) and B (minor) isomers of the rat cytochrome in the oxidized form. The A/B ratio for the rat cytochrome is 1.5 : 1, despite the very high sequence similarity (93%) to the rabbit protein. This comparison has provided insights into the factors determining the distribution in solution of the two isomers differing with respect to heme orientation. It appears that residues 23 and 74 are both important in determining this distribution, through interaction of their side chains with the prosthetic group. Hydrophobic and steric interactions are the key factors in determining the relative stability of one isomer with respect to the other. Keywords: cytochrome b5 solution structure.
Cytochrome b5 (cytb5 hereafter) is a low-spin hemoprotein which acts as an electron-transfer mediator in several redox pathways, and interacts in vivo with a number of different redox partners [1]. In the process of fatty acid desaturation, cytb5 interacts with NADH cytochrome b5 reductase [2,3]; a role of cytb5 as electron donor to cytochrome P-450 has also been proposed [4,5]. The protein is normally membrane-bound, although a soluble form is found in erythrocytes, where its physiological role is that of reducing methemoglobin [6]. The membrane-bound form may be solubilized by incubation with proteolytic enzymes, such as trypsin. The soluble fragment still binds the heme and retains protein recognition and electrontransfer capabilities [7]. The soluble fragment of cytb5 interacts in vitro with cytochrome c, the reaction between the two being fast [7]. Also thanks to the fact that the crystallographic structures of the two individual components have been available since 1971 [8,9], this complex has been an important model for the study of long-range biological electron transfer and, more generally, of the mechanisms of protein±protein recognition and interaction. The interaction between cytb5 and cytochrome c has been studied by a number of biochemical and biophysical methods (reviewed in [10]). In particular, the small size of the two proteins had allowed the investigation of their complex by NMR spectroscopy as early as 1983 [11]. Cytb5 is heterogeneous in solution, as the heme, being bound Correspondence to I. Bertini, Department of Chemistry and Centro di Risonanze Magnetiche, University of Florence, Via Luigi Sacconi 6, 50019, Sesto Fiorentino, Italy. Fax: + 39 055 4209271, Tel.: + 39 055 4209272, E-mail:
[email protected] Abbreviations: cytb5, cytochrome b5; TPPI, time proportional phase increments; pcs, pseudocontact shifts. (Received 23 August 1999, revised 25 November 1999, accepted 29 November 1999)
to the polypeptide chain only through the bonds between the iron ion and the two axial histidines, can be present in two possible conformations, related to each other by a 1808 rotation about the heme a±g meso axis [12,13]. The two conformers exchange slowly on the NMR time scale, the rate constant for the interconvertion process being of the order of 1025 s21 in the oxidized state, and about 100-fold smaller in the reduced state [13]. The two forms of cytb5 have slightly different redox potentials, that of the minor form being more negative by < 27 mV [14]. This small difference is probably not relevant for the physiological function of cytb5 [14]. It has been proposed that the differences in protein mobility of the two forms largely contribute to this difference in redox potential [15]. The molar ratio of the two conformers of microsomal rabbit cytb5 is about 5 : 1. Of those known, rat cytb5 shows the highest heterogeneity, the molar ratio of the major and minor forms being about 1.5 : 1 [16]. In all cytb5 forms, regardless of the organism from which the protein is obtained, the more abundant conformer binds the heme in the same orientation. The rat and rabbit proteins have sequence identity as high as 93%, and only two amino acid mutations occur in the environment of the heme moiety. The bovine protein, X-raycrystallographic [17,18] and solution [19] structures of which are available, has a major/minor form molar ratio of 9 : 1 [13], and shows 91% sequence identity with rabbit microsomal cytb5 and 94% sequence identity with rat microsomal cytb5. To determine why such large variations in the ratio between the two forms occur on only modest changes in the primary sequence, and their structural determinants, it is necessary to compare the structures, solved to a comparable degree of accuracy and precision, of cytb5 from at least two organisms with a sizable difference in the conformer distribution. A comparison of the structures of the minor and major form is needed as well. It should be kept in mind that the structures of
756 L. Banci et al. (Eur. J. Biochem. 267)
two proteins with a difference in sequence identity of less than 10% are not necessarily significantly different, unless such differences involve substitutions between amino acids with greatly differing side chain volume and/or chemicophysical properties. So far, it has not been possible to characterize by X-ray crystallography the structure of the minor form of cytb5, and thus only the structures of the major form of bovine [17,18] and rat outer mitochondrial membrane cytb5 [20] are available from the PDB [21]. Indeed, although rat cytb5 has been crystallized, it has not been possible to resolve the details of the structures of both different forms by X-ray crystallographic methods, because of the limited resolution achieved [20]. On the other hand, the structure of both the major [22±25] and minor forms [15,25] of cytb5 in solution has been characterized by NMR spectroscopy on the rat microsomal protein, in both oxidation states. To perform a meaningful comparison, these structures should be compared with the NMR structure of a cytb5 with a very high major/minor form ratio. For this reason, we have solved and report here the solution structure of rabbit cytb5, which has a major/minor form ratio of 5 : 1.
M AT E R I A L S A N D M E T H O D S Sample preparation Cytb5 was isolated from Escherichia coli strain TOPP2, harboring the plasmid pKK223 carrying the gene coding for the 98-amino acid polypeptide corresponding to the soluble portion of microsomal rabbit cytb5 (generously provided by A. W. Steggles, Northeastern Ohio Universities College of Medicine, OH, USA), following the procedure previously reported for rat cytb5 [26]. To prepare the 15 N-labeled samples, E. coli cells were transformed with the plasmid and plated on to Luria±Bertani agarose containing 100 mg´mL21 ampicillin. A 10 mL volume of Luria±Bertani medium containing 100 mg´mL21 ampicillin was inoculated from a single colony, and cultured overnight at 37 8C with shaking. The inoculum was then transferred to a 2-L flask containing 1 L minimal medium with 50 mg´mL21 ampicillin. The culture was grown with shaking at 37 8C up to an A600 of about 0.8, and then induced with 0.5 mm isopropyl b-d-thiogalactoside. The culture was harvested by centrifugation after 15 h from induction. The protein was extracted from the E. coli cells as previously described [26]. The minimal medium used for cultures consisted of M9 salts supplemented with MgSO4, trace metal and vitamin solutions. The nitrogen source was (15NH4)2SO4 (1.2 g´L21), and the carbon source was glycerol (1 g´L21 ). NMR samples were < 2±3 mm in protein, in 1 mm phosphate buffer, pH 7.0. The pH of protein samples was adjusted by addition of small volumes of concentrated solution of NaOH or H3PO4. NMR spectroscopy The NMR spectra were recorded on a Bruker AMX 600 or on an AVANCE 800 spectrometer operating at a proton Larmor frequency of 600.13 and 800.13 MHz, respectively. A tripleresonance 5-mm probe has been used on both spectrometers. All experiments were performed at 298 K. Three-dimensional (3D) TOCSY-15N HMQC [27] and NOESY-15N HMQC [28] experiments were recorded in H2O solution with 1024(1H) 100(15N) 512(1H) data points. In these experiments the delay between the 1H 908 pulse after the mixing period and the first subsequent 15N 908 pulse was set to
q FEBS 2000
5.5 ms (< 1/2JNH), the mixing time was 80 ms for the TOCSY-15N HMQC and 100 ms for the NOESY-15N HMQC, and the acquisition was carried out by positioning the carrier frequency in the center of the amide proton region at 7.35 p.p.m. Spectral windows of 12.0, 26.0 and 6.5 p.p.m. were used, respectively, for the 1 H and 15 N indirect dimensions and 1H direct dimension. A two-dimensional (2D) NOESY and a 2D clean-TOCSY maps were recorded at 298 K both in D2O (90%D2O/10%H2O) and H2O. The time proportional phase increment (TPPI) NOESY [29,30] spectrum was recorded with a recycle time of 800 ms and a mixing time of 100 ms. Analogously, cleanTOCSY [31,32] experiments were recorded with a recycle time of 800 ms and a spin lock time of 90 ms. Both maps were recorded on a 14-p.p.m. spectral width. To detect connectivities among hyperfine shifted signals, a 2D NOESY experiment [29,30] with a spectral width of 90 and 15 p.p.m. in the two frequency dimensions, with a recycle time of 450 ms, and with 40 ms of mixing time was acquired. In addition, 2D NOESY [29,30] (on both the H2O and on the D2O samples) and TOCSY [31,32] spectra were acquired with a mixing time of 100 ms and spin-lock time of 70 ms, respectively, and a recycle time of 900 ms. All data consisted of 4K data points in the acquisition dimension and of 1K experiments in the indirect dimension. Connectivities involving the hyperfine-shifted broad signals of the iron axial ligands were detected through one-dimensional NOEs, using the reported methodology [33], by irradiating the signals at 13.1, 10.5, 10.0 and 214.0 p.p.m. Detection of broad signals was enhanced by acquiring spectra with the SuperWEFT pulse sequence [34,35], using very short recycle times. Raw data were multiplied in both dimensions by a pure cosine-squared bell window function and Fourier-transformed to obtain 2048 2048 real data points. A polynomial baseline correction was applied in both directions. All NOESY spectra (2D and 3D) were acquired at 800 MHz, whereas all TOCSY spectra (2D and 3D) were acquired at 600 MHz. For all the experiments, quadrature detection in the indirect dimensions was performed in the TPPI mode [30]. Water suppression was achieved through the WATERGATE sequence [36], except in the NOESY experiments performed to detect connectivities between paramagnetically shifted resonances where presaturation was used and in one-dimensional NOE experiments, where the SuperWEFT sequence [34] was used. All spectra were processed using the standard Bruker software (xwinnmr) and analyzed on IBM RISC 6000 computers with the program xeasy [37]. Structure calculations The volumes of the NOESY cross-peaks between assigned resonances were manually integrated, using the elliptical integration routine implemented in the program xeasy. Dipolar connectivities were taken from the 2D NOESY map acquired on a small spectral window in both dimensions and with a low repetition rate and from the 2D NOESY map acquired on a large spectral window in both dimensions and with a high repetition rate. The cross-peak intensities in the latter map were scaled to those in the former referring to a few cross-peaks with volumes that could be accurately measured in both spectra. NOESY cross-peak intensities were converted into upper limits for interatomic distances, following the methodology of the program caliba [38]. Stereospecific assignments for geminal protons were obtained with the program glomsa [38].
q FEBS 2000 3
NMR structure of rabbit ferricytochrome b5 (Eur. J. Biochem. 267) 757
JHNHa coupling constants were converted into constraints for the backbone torsion angle f, by means of the appropriate Karplus curve [39]. Ambiguity between different angles corresponding to the same value of the 3JHNHa coupling constant were resolved by looking at structures obtained from pseudyana [40] calculations run without the ambiguous torsion angle constraints. All angles were given a ^ 208 uncertainty. Pseudocontact shifts (pcs hereafter) were employed as additional constraints for the structure calculations [41±43]. Pcs values were obtained by subtracting from the chemical shifts measured in the oxidized form of rabbit cytb5 the chemical shifts measured for the diamagnetic major form of rat cytb5. No pcs were introduced for the residues which are different in the two sequences, and for the neighboring residues. A tolerance of 10% of the estimated pcs was used in the pseudyana calculations [40] (see later) with a minimum value of 0.5 p.p.m. for all protons. The hyperfine shifts of the heme and the axial heme ligands (His39 and His63) were not included in the calculations because they experience a nonnegligible contact shift. A total of 246 pcs constraints was used. All the constraints used are reported in the Supplementary material. Structure calculations were performed using pseudyana [40], which is a modified version of the program dyana [44] adapted to include pcs as additional restraints. A preliminary family of 20 conformers obtained using only the NOE constraints was used as an input model to the program fantasian [45,46] to provide the initial values of Dxax and Dxrh. These calculations were performed for each of the 20 preliminary structures, and the average values of Dxax and Dxrh were used as starting values in the pseudyana structure calculations. The pseudyana protocol needs only the initial values of the magnetic susceptibility anisotropy tensor and not its direction cosines, i.e. no specific initial orientation is provided for the tensor. A special residue, called LTNS, formed by dummy atoms is used to define the position of the metal center and the directions of the magnetic susceptibility tensor in the dihedral angle coordinate space. The dummy atoms of this residue have their van der Waals radii set to zero so that it can freely penetrate into the protein. An upper distance limit of 0.02 nm was set between the pseudoatom defining the metal ion and the iron atom. The location of this residue and the tensor orientation were optimized during the structure calculations. After each cycle of structure calculations, the magnetic anisotropy parameters were re-evaluated and used as input for the following calculation until the final values did not deviate more than 5% from the initial ones. Three cycles were needed to reach convergence. The heme group was included in the calculations by defining a new residue in the amino acid library according to the previously described procedure [23]. The two axial ligands (His39 and His63) are linked to the iron atom through upper distance limits of 0.210 nm with the N:2 atom. This approach does not impose any fixed orientation of the axial ligands with respect to the heme. In all structure calculations, the relative weight of all classes of constraints (NOE, pcs and J couplings) was kept equal to the default pseudyana values. A total of 150 random conformers were annealed in 12 000 steps using the above constraints. The 40 conformers with the lowest target function constitute the final family. Restrained energy minimization was then applied to each member of the family using the amber 5.0 package [47]. The distance constraints were applied within the molecular mechanics and dynamics module of sander and the pcs were
included as constraints by means of the module pcshifts [46]. The force field parameters for the heme and its ligands, as well as the overall calculation procedure, were set up as previously reported for similar systems [23,48]. The initial values of the magnetic susceptibility anisotropy used as input in pseudorem were the values on the final pseudyana family. On the final pseudorem family the magnetic susceptibility anisotropy parameters deviate less than 5% from their initial values (i.e. before energy minimization). The program corma [49], which is based on relaxation matrix calculations, was used to back-calculate the NOESY cross-peaks and to check the validity of the structure. It has also been used to locate a few more cross-peaks between already assigned resonances. The quality of the structure was evaluated in terms of deviations from ideal bond lengths and bond angles and through Ramachandran plots obtained using the programs procheck [50] and procheck-nmr [51]. Structure calculations and analyses were performed on an IBM SP02 parallel computer.
R E S U LT S A N D D I S C U S S I O N Sequence-specific assignment Extensive assignments of oxidized cytb5 from different organisms are available in the literature, both for the resonances of the polypeptide chain falling in the diamagnetic range [52±54] and for hyperfine shifted signals of the heme moiety and of the axial ligands [55±58]. The present assignment (data not shown) of rabbit cytb5 is consistent with those already reported. Secondary structure Figure 1 shows the short and medium range NOEs observed for the backbone and b protons in the various NOESY maps. All the backbone proton±backbone proton NOEs are reported in Fig. 2. From these two figures it is evident that the folding of cytb5 is characterized by the presence of a large amount of secondary structure motives (i.e. helices and b-strands). Sequential NH-NH connectivities for stretches longer than two amino acids have been observed for residues 6±16, 18±21, 25±27, 34±39, 42±53, 54±75, 76±80, 82±87. Three prolines (residues 40, 81, and 90) are present in the sequence, and break the sequential NH-NH connectivities. Helical structures, characterized by strong sequential NH-NH and medium-range Ha-NH(i, i + 3), Ha-Hb(i, i + 3), and Ha-NH(i, i + 4) NOEs, are present in the segments 8±15, 32±39, 43±50, 55±62, 64±74, 82±85 (Fig. 1). The presence of an antiparallel b-sheet formed by three strands spanning residues 21±25, 28±33, and 74±79 is suggested by the detection of typical long-range backbone NOE patterns, as can be seen from Fig. 2. Moreover, NOE patterns pointing to the presence of two additional parallel b-strands involving residues 5±7 (parallel to the strand 74±79) and 50±55 (parallel to the strand 21±25) were observed (Fig. 2). Solution structure calculations A total of 1908 NOESY experimental constraints were obtained, which were transformed into upper distance limits with the program caliba [38]. The best calibration of observed intensities was found to be inversely proportional to the fifth or sixth power of the proton±proton distances, depending on the
758 L. Banci et al. (Eur. J. Biochem. 267)
q FEBS 2000
Fig. 1. Schematic representation of the sequential and medium range NOE connectivities involving NH, Ha and Hb protons. The thickness of the bar indicates the NOEs intensity.
calibration class of NOE constraints. Forty diastereotopic protons were stereospecifically assigned with glomsa [38]. Of the total 1908 experimental NOEs (data not shown), 1469 were found to be relevant (i.e. corresponding to a pair of protons with the distance not fixed) and therefore used in the dyana calculations (corresponding to an average of 21.7 experimental constraints and 16.7 meaningful NOEs per assigned residue). The number of experimental NOE constraints per residue is shown in the bottom panel of Fig. 3. The structure was also calculated using pcs as further constraints (see Materials and methods). Pcs are defined as the difference between the actual shift and the shifts experienced by the same nucleus in the absence of unpaired electron (provided that the Fermi contribution to the shift is zero, see below). Pcs were used within the point dipole approximation, i.e. where the electron is considered localized on the iron atom. Pcs values are given by [59]: ÿ 3 ÿ 1 dpcs Dxax 3n2i 2 1 Dxrh l 2i 2 m2i
1 3 2 12pr i where Dxax and Dxrh are the axial and the rhombic anisotropies of the magnetic susceptibility tensor, ri is the distance of the nucleus i from the metal ion, and li , mi and ni are the direction
cosines of the position vector of atom i with respect to the orthogonal reference system formed by the principal axes of the magnetic susceptibility tensor. The pcs constraints used are reported in the Supplementary material. In early studies, the magnetic anisotropy tensor was determined from pcs by fitting the experimental data to Eqn (1) using an available 3D structure of the system [60±65]. Sometimes structural information could be obtained [66±68]. In 1996 a protocol was developed to use pcs values as constraints, together with NOE and 3J couplings, directly in solution structure calculations [45]. Critical analyses of this and related protocols are available [40,46,69±72]. Pcs values are easily determined for the nuclei of residues not directly bound to the metal ion, for which the Fermi contribution to the hyperfine shift is negligible. Thus, the protons of the two axial histidines and of the heme are not considered. Two points should be kept in mind in this treatment: (a) the electron is indeed delocalized on the heme moiety and on the axial ligands, thus undermining the validity of Eqn (1) (see below); (b) the shifts for the species without unpaired electrons (diamagnetic shifts) can only be estimated. Point (a) is overcome by introducing a tolerance proportional to the actual pcs value. Indeed, larger pcs values, which are experienced by the protons
q FEBS 2000
NMR structure of rabbit ferricytochrome b5 (Eur. J. Biochem. 267) 759
Fig. 2. Diagonal plot of the NOEs observed between the backbone protons.
closer to the paramagnetic center and thus more affected by electron delocalization, are given a larger indetermination. As far as point (b) is concerned, the diamagnetic chemical shift can be calculated from the structure obtained from NOEs or from the shifts of the reduced diamagnetic protein. In the latter case, it is assumed that the oxidized and the reduced species do not differ in structure enough to provide a difference in the diamagnetic chemical shift larger than the tolerance given to pcs. In the present case, the diamagnetic chemical shifts were taken from the reduced rat species. This procedure is justified by the extreme similarity between the chemical shifts measured for reduced cytb5 in different species (e.g. calf, pig, rat), with the exception of residues in which the side chain differs in the various sequences [73,74]. No constraints were introduced for the residues that are different in the rabbit and rat sequences, and for their neighboring residues. A tolerance equal to 10% of the observed pseudocontact shift was given to all pcs values (see point a above). A minimal value of the tolerance of 0.5 p.p.m. was imposed to account for possible small structural rearrangements occurring far from the mutation sites. The 40 conformers with lower target function constituted the final family describing the solution structure. The family has rmsd values (for residues 4±84) to the mean structure of 0.061 ^ 0.010 nm for the backbone and 0.112 ^ 0.013 nm for the heavy atoms. The average total target function values for the above 40 structures was 0.0093 ^ 0.0013 nm2. The 40 conformers obtained from dyana calculations were subjected to restrained energy minimization (pseudorem) [46,47]. The resulting family (schematically shown in Fig. 4) has rmsd values (for residues 4±84) of 0.059 ^ 0.011 nm and 0.112 ^ 0.011 nm with respect to the mean structure. NOEs have an average penalty function of 66 kJ´mol21
(corresponding to a dyana target function of about 0.005 nm2 ). The rmsd values from the average structure per residue are reported in the top panel of Fig. 3. It can be seen that, besides the N-terminal and C-terminal regions, the backbone rmsd is satisfactorily low for almost all residues, including those in the proximity of the paramagnetic center. The only exception is residues 17±19, which comprise the unassigned residue Ser18. All the following analyses will refer to the well-defined portion of the polypeptide chain constituted by residues 4±84. Secondary-structure elements were identified by using the programs procheck [50] and procheck-nmr [51]. The analysis of the family of conformers with the latter program highlights the following elements of secondary structure: residues 8±13, 33±39, 42±49, 52±62, 64±74, 82±84 form helical structures, residue 21±25, 26±32, and 75±79 form a three-stranded b-sheet. A fourth strand of the b-sheet is formed by residues 5±7 in 30% of the structures. In all, 71.7% of the residues in the well defined region 4±84 belong to one of the most favored regions, whereas 23.0% are found in an allowed region and 4.8% in a generously allowed region. Less than 1 residue per conformer is found in a disallowed region. In the mean structure, 80.3% of the residues are found in the most favored regions of the Ramachandran plot, 18.3% in the allowed regions, and only one residue is in a generously allowed region. No residues were found in the disallowed regions. For the mean structure, the following residues were found to form a-helices: 8±13, 42±50, 54±62, 64±74. A 310 helix is reported for residues 33±39 and 80±84. Residues 21±23, 28±32 and 75±79 form a three-stranded b-sheet. Table 1 reports calculation statistics and the structure quality analysis for the final family.
760 L. Banci et al. (Eur. J. Biochem. 267)
q FEBS 2000
Fig. 3. The number of observed experimental NOEs per residue (bottom) is compared with the rmsd values (top) for the backbone (W) and all heavy atoms (B). Black bars indicate long range NOEs, grey bars medium range NOEs, light grey bars sequential NOEs, and white bars intraresidue NOEs. The data for residue 63 includes also the heme moiety.
The magnetic susceptibility tensor The final Dxax and Dxrh values are (2.66 ^ 0.09) 10232 m3 and (20.91 ^ 0.11) 10232 m3, respectively. The z-axis of the magnetic susceptibility tensor makes an angle of less than 58 with the normal to the heme plane, whereas the x-axis of the tensor makes an angle of < 108 with the a±g meso direction and the y-axis makes an angle of < 78 with the b±d meso direction. These values are essentially identical (within experimental error) with those reported for the solution structure of the A form of oxidized rat cytb5 [23], showing that the electronic structure of the iron and its ligands is not appreciably perturbed by the mutations occurring in the polypeptide chain. Thus, it seems that there is no close relationship between the distribution of conformers in cytb5 and the electronic structure of the metal site. This is consistent with the idea that the electronic structure of the heme, being central to the rate and efficiency of electron transfer to the physiological partners, should be maintained in homologus cytb5 from different, but evolutionarily similar, organisms. The
orientation of the in-plane axes of the magnetic susceptibility tensor is known to be essentially dependent on the orientation of the iron axial ligands, i.e. in this case, on the orientation of the imidazole planes of the axial histidines [58,75,76]. If the two planes are parallel, and their normal is aligned along one metal±pyrrole nitrogen direction, then the y-axis of the tensor will be along the same direction. Note that this direction is orthogonal to the normal plane of the p system of the imidazoles. A rotation of both planes of a given angle with respect to the metal±pyrrole nitrogen direction will cause a rotation of the same angle of the y-axis of the tensor in the opposite direction. Furthermore, a rotation of a given angle of one plane with respect to the other would cause a rotation of the tensor y-axis of half that angle in the opposite direction (note that this is true only when the p interactions between each of the two axial ligands and the iron ion are equally important). These two effects add up: in practice, the rotation of the y-axis of the tensor with respect to a given Fe±pyrrole nitrogen direction is equal in magnitude, but opposite, to the rotation of the bisector of the normals to the two imidazole planes with
q FEBS 2000
NMR structure of rabbit ferricytochrome b5 (Eur. J. Biochem. 267) 761
Fig. 4. Display of the backbone of residues 4±84 of the final pseudorem family of conformers as a tube with variable radius, proportional to the backbone rmsd of each residue. Elements of secondary structure, as determined on the final family of conformers, are highlighted: helices are in red, b-strands are in light blue. The heme moiety and the side chains of the axial histidines are shown as well. This figure was prepared with molmol [82].
respect to the same Fe±pyrrole nitrogen direction. It should be noted that, when the two planes are orthogonal to each other, the symmetry of the system is close to C4, and thus there is no sizable rhombicity. In the present case, the normal to the plane of His39 makes an angle of 408 with the Fe±pyrrole I nitrogen direction, while the normal to the plane of His63 makes an angle of 208 with the same direction (the indetermination on the observed angles are of the order of 5±108, as estimated from the spread of the family). These values are essentially identical with those observed in the solution structure of the A form of the rat protein and in the solid-state structure of the bovine protein. The two rotations are in the same direction, bringing the normal to the His planes closer to the the a±g meso direction. It is thus expected that the y-axis of the magnetic susceptibility tensor makes an angle of 308 with the Fe±pyrrole I nitrogen direction, moving towards the b±d meso direction, which is in excellent agreement with the observed average value of 388. Comparison with the structures of the A and B forms of rat cytb5 The rmsd between the backbone atoms (for the well-defined 4±84 region) of the present energy-minimized average solution structure and the energy-minimized average solution structures of the A and B forms of rat cytb5 is 0.094 and 0.090 nm, respectively. These values are, within the spread of each family of conformers, essentially equal to the rmsd value between the average structures of the A and B forms of oxidized rat cytb5 (0.091 nm), and marginally larger than the rmsd reported for the A and B forms of the reduced protein (0.052 nm) [25]. They are also comparable with the rmsd values observed between the above mentioned solution structures and the X-ray-crystallographic structure of bovine cytb5, which vary between 0.09 and 0.11 nm. The overall fold of cytb5 is thus very well maintained in all the different species, and is not sizably affected when passing from the crystal to solution. Within this frame it is sensible to analyze in detail the conformational differences occurring at and in proximity to the residues with side chains
that differ in rabbit cytb5 with respect to the rat protein. We will focus only on the two mutations (residues 23 and 74) that are close (i.e. in van der Waals contact) to the heme moiety. Indeed, the other mutations are far from the cofactor (1.7 nm or more), and, given the fact that the secondary and tertiary structure of the protein accommodate these replacements without any significant perturbation, it is very unlikely that they can play a role in determining the distribution in solution of the two different cytb5 conformers. These mutations involve mainly solvent accessible residues, which are known to be most often replaced in families of homologous proteins [77]. Instead, residues 23 and 74 belong to the hydrophobic core of cytb5. Table 2 reports the distances between some atoms of the side chain and the backbone of residue 23 and the CMC (which is the carbon of methyl 3) and CAB (which is the a-carbon of vinyl 2) carbons of the porphyrin measured in the various solution structures of oxidized cytb5 available. The position of the latter two atoms is interconverted upon a 1808 rotation of the heme around the a±g meso axis, i.e. when passing from the major to the minor form of cytb5. Methyl 3 is the heme ring substituent closest to the side chain of Val23. Indeed, its distance from the methyl groups of the side chain in the present structure is smaller than 0.5 nm. As shown in Table 2, both the side chain and the backbone of Val23 in the minor form of rat cytb5 move away from the heme moiety by 0.04 nm with respect to the major form in order to accommodate the bulkier vinyl group. In rabbit cytb5, the larger side chain of Leu with respect to Val imposes that, in the major conformation, in which the less bulky methyl 3 faces residue 23, the backbone of the latter is further apart from the heme with respect to the rat protein (Fig. 5). This is also in agreement with the observation that the NOEs between the backbone protons of residue 23 with residues 50±52, which lie close to it, on the opposite side with respect to the heme are stronger in the rabbit than in the rat protein (not shown). It is thus reasonable to suppose that a further displacement of the backbone of Leu23 in rabbit cytb5, which would be needed to accommodate the vinyl group in the minor conformation, is energetically unfavorable, also because it would involve an, at least partial, repackaging of the b-sheet behind the heme moiety. This factor contributes to the increase in the difference in energy between the two forms of cytb5, which provides a molecular explanation for the observation that Leu to Val replacement in bovine cytb5 changes the form A/form B ratio from 9 : 1 to 4 : 1 [78]. It is also interesting to note that the rearrangement of the position of Val23 in rat cytb5 occurring upon rotation of the heme group prevents the formation of cavities between the hydrophobic part of the heme (i.e. the side of vinyl 2 and methyl 3) and the polypeptide chain, which aligns mostly hydrophobic residues in that part of the structure. The latter pack against the moiety, forming several hydrophobic contacts, which would be weakened or even lost if a cavity was formed upon rotation of the heme around the a±g meso axis. This would impose an energetic requirement greater than that needed to rearrange the local structure. The second residue that is in proximity to the heme moiety and has a different side chain in rat and rabbit cytb5 is residue 74. Rat cytb5 has a Tyr at this position, while rabbit cytb5 has a Phe. In the present solution structure, the side chain of Phe74 is somewhat closer to the heme moiety than the side chain of Tyr74 in the solution structure of both forms of the rat protein (Fig. 5). In contrast, the position of the Tyr74 side chain in the structures of the A and B form of rat cytb5 is not remarkably different (Fig. 5). The different positions of residue 74 in the rat and rabbit proteins is supported by differences in the observed
762 L. Banci et al. (Eur. J. Biochem. 267)
q FEBS 2000
Table 1. Calculation statistics and structural analysis for the final pseudorem family of oxidized rabbit cytochrome b5. Total number of NOE constraints Intraresidue Sequential Medium rangea Long range Total
305 340 353 471 1469
RMS violation per NOE constraint (average over the family) Intraresidue Sequential Medium rangea Long range Total
0.00271 ^ 0.00027 0.00150 ^ 0.00015 0.00134 ^ 0.00024 0.00119 ^ 0.00021 0.00172 ^ 0.00013
Average number of NOE violations greater than 0.005 nm per structure Intraresidue Sequential Medium rangea Long range Total
15.4 8.8 7.6 7.9 39.7
^ ^ ^ ^ ^
2.7 1.4 2.1 2.2 4.2
Average no. of NOE violations larger than 0.03 nm
0.0
Average no. of f dihedral angle violations larger than 58
0.0
Largest residual NOE violation
0.024 nm
RMS violation per pseudocontact shift (average over the family)
0.0059 ^ 0.0006 p.p.m.
Average NOE and pcs penalty function
66 ^ 8 kJ´mol21
PROCHECK-NMR analysisb Residues in most favorable regions Residues in allowed regions Residues in generously regions Residues in disallowed regions
71.7% 23.0% 4.8% 0.5%
a
Medium range distance constraints are those between residues (i, i + 2) (i, i + 3) (i, i + 4) and (i, i + 5); analysis for residues 4±84
NOEs. In particular, a NOE of medium intensity between the Hz proton of Phe74 and methyl 1 of the heme moiety is detected in the NOESY spectrum of rabbit cytb5. This NOE is not consistent with a conformation such as that observed for Tyr74 in rat cytb5 or that of Phe74 in the X-ray crystallographic structure of the bovine protein, supporting the observed conformational change. The substituent of the porphyrin closest to this side chain is methyl 1, which, in the B form of rat cytb5, is `replaced' by vinyl 4. Thus, when going from the A conformation to the B conformation, there is an increase in the steric hindrance of the heme moiety in the proximity of residue 74. As the side chain of the latter is quite close to the heme in the rabbit protein, whereas it is further away from it in the rat protein, it is reasonable to think that the B conformation is energetically less favorable in the former system than in the latter, in agreement with experimental observations. The difference in the position of the Tyr side chain with respect to the Phe is due to the presence of the hydroxy group in the former amino acid. Indeed, the Tyr side chain in the rat structures is folded in a way such that the solvent accessibility of the hydroxy group is enhanced with respect to what it would be in the conformation observed in the rabbit protein. This may be because the hydroxy group of the Tyr is capable of forming hydrogen bonds with the solvent, and thus there is an energetic contribution favorable to increasing its solvent accessibility. On the other hand, the more hydrophobic side chain of the Phe cannot give rise to energetically favorable interactions with the solvent, and consequently it adopts a conformation that
nm nm nm nm nm
b
As it results from the Ramachandran plot
maximizes its interaction with the hydrophobic part of the porphyrin moiety. Interestingly, in the crystal structure of bovine cytb5, this residue is found in a conformation closer to that observed in the rat protein with respect to that found for rabbit cytb5. It thus appears that a conformational rearrangement of the side chain of Phe74 occurs on changing from the crystal to solution. The above explanation for the role of Phe74 in modulating the isomer ratio in cytb5 is also consistent with the observation that substitution of Phe74 with other bulky hydrophobic amino acids, such as Leu, does not alter significantly the form A/form B ratio [78]. This finding again points to the importance of hydrophobic interactions between the heme moiety and the side chain of residue 74. It should be mentioned that, as can also be noted from Fig. 5, there is no significant displacement of the heme from the binding pocket in the B form of oxidized rat cytb5 with respect to the A form. This is at variance with the results of a comparison of the reduced forms, where a 0.09-nm displacement was claimed [25]. A small rotation of the heme about an axis essentially normal to the heme plane can be observed in Fig. 5 for form B of rat cytb5, which however, is not supported by significant differences in the observed NOEs. The position of the heme moiety in rabbit cytb5 is essentially identical with that of the A form of rat cytb5 (Fig. 5). The chemical shifts of the propionate protons are very similar in oxidized rat and rabbit cytb5. Given the fact that the magnetic susceptibility tensor is essentially identical in the two systems, this implies that the Fermi contribution to the hyperfine shift is very similar
q FEBS 2000
NMR structure of rabbit ferricytochrome b5 (Eur. J. Biochem. 267) 763
Table 2. Distances between the heavy atoms of residue 23 and the CMC (rabbit cytb5 and form A of rat cytb5) or CAB (form B of rat cytb5) atom of the heme moiety. The CMC atom is the carbon atom of heme methyl 3; the CAB atom is the a carbon of vinyl 2. For rabbit cytb5 and the A form of rat cytb5, the distance is referred to the CMC atom, whereas for the B form of rat cytb5 the distance is referred to the CAB atom. CMC/CAB distance (nm) Protein
CB 23
CA 23
C 23
N 23
Rabbit cytb5 (mean structure) Rat cytb5 (form A, mean structure) Rat cytb5 (form B, mean structure) Rabbit cytb5 (family) Rat cytb5 (form A, family) Rat cytb5 (form B, family)
0.59 0.52 0.56 0.55±0.62 0.48±0.57 0.49±0.59
0.73 0.67 0.71 0.70±0.76 0.64±0.72 0.65±0.74
0.77 0.72 0.76 0.74±0.80 0.70±0.78 0.70±0.78
0.80 0.76 0.79 0.78±0.84 0.72±0.80 0.71±0.81
in the two systems. As the latter contribution depends on the conformation of the propionates with respect to the heme moiety [79], it can be concluded that the latter is the same in rat and rabbit cytb5. This finding is important in view of the fact that heme propionates play a subtle role in tuning the interaction of the polypeptide chain with the heme cofactor [80]. Interpretation of data on synthetic porphyrins The porphyrin naturally occurring in cytb5 is protoporphyrin IX. It has been shown by NMR that cytb5 can bind a number of differently substituted iron porphyrins, and that two isomers may be obtained also with these synthetic porphyrins, still differing for a 1808 rotation around the a±g meso axis [16,81]. The A/B ratio was found to be highly dependent on the nature of the substituents of the porphyrin [16,81]. These investigations were performed on the rat and bovine protein. As the amino acid sequence in the regions constituting the heme pocket in the bovine and in the present cytb5 are identical, it is reasonable to discuss the above data in the light of the present structure. By comparing the data relative to a symmetric porphyrin in which all groups at positions 1, 2, 3 and 4 are methyls (for which the A/B ratio is obviously 1 : 1), with the data relative to porphyrins in which there is either a hydrogen or a vinyl at
Fig. 5. Close-up of the superposition of the average solution structures of rabbit cytb5 (blue) and the forms A (red) and B (green) of rat cytb5. All heavy atoms of residues 23 and 74 are shown, together with the backbone atoms of residues 21±25 and 72±76, and the heavy atoms of the heme moiety and of the imidazole rings of the axial histidines.
position 2, it is found that for the bovine protein the presence of a bulkier group (with respect to methyl) at position 2 makes the A isomer strongly favored, whereas the presence of a smaller group leaves the A/B ratio very close to 1. In this protein, a cavity exists to allocate the naturally occurring vinyl 2, which is apparently tolerant to reductions in the volume of the substituent at position 2. On the other hand, in the region of the structure where the vinyl is located in the B form, there is room only to accommodate a methyl group, and the structural rearrangement necessary to accommodate the bulkier vinyl group is energetically unfavorable. A smaller group, however, can still fit satisfactorily into the cavity, thereby justifying the fact that the A/B ratio for the porphyrin with a hydrogen at position 2 is 1.2 : 1. It is perhaps surprising that the conformation in which, with respect to the native system, the change in the volume occupied by the porphyrin due to replacement of the vinyl with a hydrogen is entirely located in a single region of the structure, rather than distributed over the two cavities, is preferred. The observation that the A/B ratio is higher than 1 may suggest that the cavity accommodating vinyl 2 in the native protein can compensate for a reduction in the volume of substituent 2 at an energetic cost lower than that required for the cavity accommodating methyl 3. In the rat protein, the A/B ratio for the porphyrin with a proton in position 2 is 1.8, indicating that this tendency is enhanced, probably because of the reduction in the volume of the side
764 L. Banci et al. (Eur. J. Biochem. 267)
chain of the residues forming the cavity accommodating methyl 3 as the result of the Leu23!Val substitution. The latter substitution makes packing around position 3 less tight, and this, coupled with the poorer capability of this region to accommodate smaller porphyrin substituents, makes the reduction in the volume of substituent 3 very unfavorable, presumably because of the weakening or loss of hydrophobic interactions that would occur. This reasoning is also in agreement with the finding that in the rat protein, for the porphyrin with a vinyl at position 2 and a methyl at position 3, the B isomer is less unfavoured with respect to the case of the bovine protein. Indeed, in the B conformation this porphyrin places a smaller residue in the cavity of position 2, which can accommodate this reduction in volume easily, and a bulkier residue in the cavity of position 3, which can accommodate it thanks to the decrease in the steric crowding of the residues constituting it in the rat with respect to the bovine protein (in which conformation A was strongly preferred, see above). Concluding remarks The solution structure of oxidized rabbit has been solved to a degree of resolution comparable with that of the structure of both forms of rat cytb5. This has allowed a detailed analysis of the factors tuning the distribution in solution of the conformers differing with respect to the orientation of the heme within the protein frame. Hydrophobic and steric interactions between the heme and the residues constituting the heme-binding pocket are key to tuning the heme distribution. The electronic properties of the metal ion are negligibly affected by the different distribution of forms A and B. Finally, the similarity of the measured chemical shifts and the observed NOEs supports the observation that the conformation of the heme propionates, apart from the disordered carboxylate moiety, is essentially the same in the rat and rabbit systems.
ACKNOWLEDGEMENTS We thank Dr A. W. Steggles for generously providing the gene encoding the soluble fragment of rabbit cytb5. We are grateful to F. Ferroni for the preparation of unlabeled samples and for the acquisition and analysis of preliminary spectra. The present investigation was supported by the European Union (TMR program, contract FMRX-CT98-0218 and Large Scale Facility Grant ERBFMGECT950033) and by MURST ex 40%.
REFERENCES 1. Mathews, F.S. (1985) The structure, function and evolution of cytochromes. Progr. Biophys. Mol. Biol. 45, 1±56. 2. Oshino, N., Imai, Y. & Sato, R. (1971) A function of cytochrome b5 in fatty acid desaturation by rat liver microsomes. J. Biochem. (Tokyo) 69, 155±167. 3. Strittmatter, P., Spatz, L., Corcoran, D., Rogers, M.J., Setlow, B. & Redline, R. (1974) Purification and properties of rat liver microsomal stearyl coenzyme A desaturase. Proc. Natl Acad. Sci. USA 71, 4565±4569. 4. Hildebrandt, A. & Estabrook, R.W. (1971) Evidence for the participation of cytochrome b5 in hepatic microsomal mixed function oxidation reactions. Arch. Biochem. Biophys. 143, 66±79. 5. Imai, Y. & Sato, R. (1977) The roles of cytochrome b5 in a reconstituted N-demethylase system containing cytochrome P-450. Biochem. Biophys. Res. Commun. 75, 420±426. 6. Hegesh, E., Hegesh, J. & Kaftory, A. (1986) Congenital methemoglobinemia with a deficiency of cytochrome b5. New Engl. J. Med. 314, 757±761.
q FEBS 2000 7. Strittmatter, P. & Ozols, J. (1966) The restricted tryptic cleavage of cytochrome b5. J. Biol. Chem. 241, 4787±4792. 8. Mathews, F.S., Levine, M. & Argos, P. (1971) The structure of calf liver Ê resolution. Nat. New Biol. 233, 15±16. cytochrome b5 at 2.8 A 9. Dickerson, R.E., Takano, T., Eisenberg, D., Kallai, O.B., Samson, L., Cooper, A. & Margoliash, E. (1971) Ferricytochrome c. I. General Ê resolution. J. Biol. features of the horse and bonito proteins at 2.8 A Chem. 246, 1511±1535. 10. Mauk, A.G., Mauk, M.R., Moore, G.R. & Northrup, S.H. (1995) Experimental and theoretical analysis of the interaction between cytochrome c and cytochrome b5. J. Bioenerg. Biomemb. 27, 311±330. 11. Eley, C.G.S. & Moore, G.R. (1983) 1H-NMR investigation of the interaction between cytochrome c and cytochrome b5. J. Biochem. (Tokyo) 215, 11±21. 12. Keller, R.M. & WuÈthrich, K. (1980) Structural study of the heme crevice in cytochrome b5 based on individual assignments of the NMR lines of the heme group and selected amino acid residues. Biochim. Biophys. Acta 621, 204±217. 13. McLachlan, S.J., La Mar, G.N., Burns, P.D., Smith, K.D. & Langry, K.C. (1986) 1H NMR assignment and the dynamics of interconversion of the isomeric forms of cytochrome b5 in solution. Biochim. Biophys. Acta 874, 274±284. 14. Walker, F.A., Emrick, D., Rivera, J.E., Hanquet, B.J. & Buttlaire, D.H. (1988) Effect of heme orientation on the reduction potential of cytochrome b5. J. Am. Chem. Soc. 110, 6234±6240. 15. Arnesano, F., Banci, L., Bertini, I., Felli, I.C. & Koulougliotis, D. (1999) Solution structure of the B form of oxidized rat microsomal cytochrome b5 and backbone dynamics via 15N rotating frame NMR relaxation measurements: biological implications. Eur. J. Biochem. 260, 347±354. 16. Lee, K.-B., La Mar, G.N., Kehres, L.A., Fujinari, E.M., Smith, K.M., Pochapsky, T.C. & Sligar, S.G. (1990) 1H-NMR study of the influence of hydrophobic contacts on protein-prosthetic group recognition in bovine and rat ferricytochrome b5. Biochemistry 29, 9623±9631. 17. Mathews, F.S., Argos, P. & Levine, M. (1972) The structure of Ê resolution. Cold Spring Harb. Symp. Quant. cytochrome b5 at 2.0 A Biol. 36, 387. 18. Durley, R.C.E. & Mathews, F.S. (1996) Refinement and structural Ê resolution. Acta analysis of bovine cytochrome b5 at 1.5 A Crystallogr. D 52, 65±72. 19. Muskett, F.W., Kelly, G.P. & Whitford, D. (1996) The solution structure of bovine ferricytochrome b5 determined using heteronuclear NMR methods. J. Mol. Biol. 258, 172±189. 20. Rodriguez Maranon, M.J., Qiu, F., Stark, R.E., White, S.P., Zhang, X., Foundling, S.I., Rodriguez, V., Schilling, C.L., Bunce, R.A. & Rivera, M. (1996) 13C Spectroscopic and X-ray crystallographic study of the role played by mitochondrial cytochrome b5 heme propionates in the electrostatic binding to cytochrome c. Biochemistry 35, 16378±16390. 21. Bernstein, F.C., Koetzle, T.F., Williams, G.J.B., Meyer, E.F. Jr, Rodgers, J.R., Kennard, O., Shimanouchi, T. & Tasumi, M. (1977) The protein data bank: a computer-based archival file for macromolecular structure. J. Mol. Biol. 112, 535±542. 22. Banci, L., Bertini, I., Ferroni, F. & Rosato, A. (1997) Solution structure of reduced microsomal cytochrome b5. Eur. J. Biochem. 249, 270±279. 23. Arnesano, F., Banci, L., Bertini, I. & Felli, I.C. (1998) The solution structure of oxidized rat microsomal cytochrome b5. Biochemistry 37, 173±184. 24. Banci, L., Bertini, I., Huber, J.G., Luchinat, C. & Rosato, A. (1998) Partial orientation of oxidized and reduced cytochrome b5 at high magnetic fields: magnetic susceptibility anisotropy contributions and consequences for protein solution structure determination. J. Am. Chem. Soc. 120, 12903±12909. 25. Dangi, B., Sarma, S., Yan, C., Banville, D.L. & Guiles, R.D. (1998) The origin of differences in the physical properties of the equilibrium forms of cytochrome b5 revealed through high-resolution nmr
q FEBS 2000
26.
27.
28. 29.
30.
31. 32.
33.
34.
35.
36. 37.
38.
39.
40.
41. 42.
43. 44. 45.
46.
NMR structure of rabbit ferricytochrome b5 (Eur. J. Biochem. 267) 765
structures and backbone dynamic analyses. Biochemistry 37, 8289±8302. von Bodman, S.B., Schulder, M.A., Jollie, D.R. & Sligar, S.G. (1986) Synthesis, bacterial expression, and mutagenesis of the gene coding for mammalian cytochrome b5. Proc. Natl Acad. Sci. USA 83, 9443±9447. Marion, D., Kay, L.E., Sparks, S.W., Torchia, D.A. & Bax, A. (1989) Three dimensional heteronuclear NMR of 15N-labeled proteins. J. Am. Chem. Soc. 111, 1515±1517. Kay, L.E., Marion, D. & Bax, A. (1989) Pratical aspects of 3D heteronuclear NMR of proteins. J. Magn. Reson. 84, 72±84. Macura, S., WuÈthrich, K. & Ernst, R.R. (1982) The relevance of J cross-peaks in two-dimensional NOE experiments of macromolecules. J. Magn. Reson. 47, 351±357. Marion, D. & WuÈthrich, K. (1983) Application of phase sensitive correlated spectroscopy (COSY) for measurements of proton-proton spin-spin coupling constants in proteins. Biochem. Biophys. Res. Commun. 113, 967±974. Bax, A. & Davis, D.G. (1985) Practical aspects of two-dimensional transverse NOE spectroscopy. J. Magn. Reson. 63, 207±213. Griesinger, C., Otting, G., WuÈthrich, K. & Ernst, R.R. (1988) Clean TOCSY for 1H spin system identification in macromolecules. J. Am. Chem. Soc. 110, 7870±7872. Banci, L., Bertini, I., Luchinat, C., Piccioli, M., Scozzafava, A. & Turano, P. (1989) 1H NOE studies on dicopper (II) dicobalt (II) superoxide dismutase. Inorg. Chem. 28, 4650±4656. Inubushi, T. & Becker, E.D. (1983) Efficient detection of paramagnetically shifted NMR resonances by optimizing the WEFT pulse sequence. J. Magn. Reson. 51, 128±133. Rigby, S.E.J., Burch, A.M. & Moore, G.R. (1991) 1H-NMR determination of the ionization constant of a carboxylic acid group of a haem protein in mixed aqueous-organic solvents using the SuperWEFT sequence. Magn. Reson. Chem. 29, 1036±1039. Piotto, M., Saudek, V. & Sklenar, V. (1992) Gradient-tailored excitation for single quantum NMR spectroscopy of aqueous solutions. J. Biomol. NMR 2, 661±666. Eccles, C., GuÈntert, P., Billeter, M. & WuÈthrich, K. (1991) Efficient analysis of protein 2D NMR spectra using the software package EASY. J. Biomol. NMR 1, 111±130. GuÈntert, P., Braun, W. & WuÈthrich, K. (1991) Efficient computation of three-dimensional protein structures in solution from nuclear magnetic resonance data using the program DIANA and the supporting programs CALIBA, HABAS and GLOMSA. J. Mol. Biol. 217, 517±530. Vuister, G.W. & Bax, A. (1993) Quantitative J correlation: a new approach for measuring homonuclear three-bond J (HNHa) coupling constants in 15N enriched proteins. J. Am. Chem. Soc. 115, 7772±7777. Banci, L., Bertini, I., Cremonini, M.A., Gori Savellini, G., Luchinat, C., WuÈthrich, K. & GuÈntert, P. (1998) PSEUDODYANA for NMR structure calculation of paramagnetic metalloproteins using torsion angle molecular dynamics. J. Biomol. NMR 12, 553±557. Bertini, I. & Luchinat, C. (1986) NMR of Paramagnetic Molecules in Biological Systems. Benjamin/Cummings, Menlo Park, CA. Bertini, I. & P.Turano (1994) The hyperfine coupling. In NMR of Paramagnetic Macromolecules. NATO ASI Series (La Mar, G.N., eds), pp. 29±54. Kluwer Academic, Dordrecht, the Netherlands. Bertini, I. & Luchinat, C. (1996). NMR of paramagnetic substances. Coord. Chem. Rev. 150, pp. 1±300. GuÈntert, P., Mumenthaler, C. & WuÈthrich, K. (1997) Torsion angle dynamics for NMR structure calculation with the new program DYANA. J. Mol. Biol. 273, 283±298. Banci, L., Bertini, I., Bren, K.L., Cremonini, M.A., Gray, H.B., Luchinat, C. & Turano, P. (1996) The use of pseudocontact shifts to refine solution structures of paramagnetic metalloproteins: Met80Ala cyano-cytochrome c as an example. J. Biol. Inorg. Chem. 1, 117±126. Banci, L., Bertini, I., Gori Savellini, G., Romagnoli, A., Turano, P., Cremonini, M.A., Luchinat, C. & Gray, H.B. (1997) The pseudocontact shifts as constraints for energy minimization and molecular
47.
48.
49. 50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
dynamic calculations on solution structures of paramagnetic metalloproteins. Proteins 29, 68±76. Pearlman, D.A., Case, D.A., Caldwell, J.W., Ross, W.S., Cheatham, T.E., Ferguson, D.M., Seibel, G.L., Singh, U.C., Weiner, P.K. & Kollman, P.A. (1997) AMBER 5.0. University of California, San Francisco. Banci, L., Bertini, I., Gray, H.B., Luchinat, C., Reddig, T., Rosato, A. & Turano, P. (1997) Solution structure of oxidized horse heart cytochrome c. Biochemistry 36, 9867±9877. Borgias, B., Thomas, P.D. & James, T.L. (1989) Complete Relaxation Matrix Analysis (CORMA). University of California, San Francisco. Laskowski, R.A., MacArthur, M.W., Moss, D.S. & Thornton, J.M. (1993) PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283±291. Laskowski, R.A., Rullmann, J.A.C., MacArthur, M.W., Kaptein, R. & Thornton, J.M. (1996) AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J. Biomol. NMR 8, 477±486. Veitch, N.C., Whitford, D. & Williams, R.J.P. (1990) An analysis of the pseudocontact shifts and their relationship to stuctural features of the redox states of cytochrome b5. FEBS Lett. 269, 297±304. Guiles, R.D., Basus, V.J., Sarma, S., Malpure, S., Fox, K.M., Kuntz, I.D. & Waskell, L. (1993) Novel heteronuclear methods of assignment transfer from a diamagnetic to a paramagnetic protein: application to rat cytochrome b5. Biochemistry 32, 8329±8340. Sarma, S., DiGate, R.J., Banville, D. & Guiles, R.D. (1996) 1H, 13C and 15 N NMR assignments and secondary structure of the paramagnetic form of rat cytochrome b5. J. Biomol. NMR 8, 171±183. Lee, K.-B., La Mar, G.N., Mansfield, K.E., Smith, K.M., Pochapsky, T.C. & Sligar, S.G. (1993) Interpretation of hyperfine shift patterns in ferricytochromes b5 in terms of angular position of the heme: a sensitive probe for peripheral heme protein interactions. Biochim. Biophys. Acta 1202, 189±199. Sun, Y.L., Wang, Y.H., Yan, M.M., Sun, B.Y., Xie, Y., Huang, Z.X., Jiang, S.K. & Wu, H.M. (1999) Structure, interaction and electron transfer between cytochrome b5, its E44A and/or E56A mutants and cytochrome c. J. Mol. Biol. 285, 347±359. Banci, L., Bertini, I., Pierattelli, R. & Vila, A.J. (1994) 1H 13C HETCOR investigations on heme-containing systems. Inorg. Chem. 33, 4338±4343. Banci, L., Pierattelli, R. & Turner, D.L. (1995) Determination of haem electronic structure in cytochrome b5 and metcyanomyoglobin. Eur. J. Biochem. 232, 522±527. Kurland, R.J. & McGarvey, B.R. (1970) Isotropic NMR shifts in transition metal complexes: calculation of the Fermi contact and pseudocontact terms. J. Magn. Reson. 2, 286±301. Feng, Y.Q., Roder, H. & Englander, S.W. (1990) Redox dependent structure change and hyperfine nuclear magnetic resonance shifts in cytochrome c. Biochemistry 29, 3494±3504. Emerson, S.D. & La Mar, G.N. (1990) NMR determination of the orientation of the magnetic susceptibility tensor in cyanometmyoglobin: a new probe of steric tilt of bound ligand. Biochemistry 29, 1556±1566. Gao, Y., Boyd, J., Pielak, G.J. & Williams, R.J.P. (1991) Comparison of reduced and oxidized yeast iso-1-cytochrome c using proton paramagnetic shifts. Biochemistry 30, 1928±1934. Rajarathnam, K., La Mar, G.N., Chiu, M.L. & Sligar, S.G. (1992) Determination of the orientation of the magnetic axes of the cyanometMb complexes of point mutants of myoglobin by solution 1H NMR: influence of HisE7-Gly and Arg CD3-Gly substitutions. J. Am. Chem. Soc. 114, 9048±9058. La Mar, G.N., Chen, Z.G., Vyas, K. & McPherson, A.D. (1995) An interpretative basis of the hyperfine shifts in cyanide-inhibited horseradish peroxidase on the magnetic axes and ligand tilt. Influence of substrate binding and extension to other peroxidases. J. Am. Chem. Soc. 117, 411±419. Banci, L., Bertini, I., Pierattelli, R., Tien, M. & Vila, A.J. (1995) Factoring of the hyperfine shifts in the cyanide adduct of lignin
766 L. Banci et al. (Eur. J. Biochem. 267)
66.
67.
68.
69. 70.
71.
72.
73.
74.
75.
peroxidase from P. chrysosporium. J. Am. Chem. Soc. 117, 8659±8667. Barry, C.D., North, A.C.T., Glasel, J.A., Williams, R.J.P. & Xavier, A.V. (1971) Quantitative determination of mononucleotide conformations in solution using lanthanide ion shift and broading NMR probes. Nature (London) 232, 236±245. Barry, C.D., Glasel, J.A., Williams, R.J.P. & Xavier, A.V. (1974) Quantitative determination of conformations of flexible molecules in solution using lanthanide ions as nuclear magnetic resonance probes: application to adenosine-5 0 -monophosphate. J. Mol. Biol. 84, 471±490. Barry, C.D., Martin, D.R., Williams, R.J.P. & Xavier, A.V. (1974) Quantitative determination of the conformation of cyclic 3 0 ,5 0 -adenosine monophosphate in solution using lanthanide ions as nuclear magnetic resonance probes. J. Mol. Biol. 84, 491±502. Bertini, I., Luchinat, C. & Rosato, A. (1996) The solution structure of paramagnetic metalloproteins. Progr. Biophys. Mol. Biol. 66, 43±80. Turner, D.L., Brennan, L., Chamberlin, S.G., Louro, R.O. & Xavier, A.V. (1998) Determination of solution structures of paramagnetic proteins by NMR. Eur. Biophys. J. 27, 367±375. Tu, K. & Gochin, M. (1999) Structure determination by restrained molecular dynamics using NMR pseudocontact shifts as experimentally determined constraints. J. Am. Chem. Soc. 121, 9276±9285. Banci, L., I.Bertini, C., Luchinat & P., Turano (1999) Solution structures of hemoproteins. In The Porphyrin Handbook (Kadish, K.M., Smith, K.M. & Guilard, R., eds), pp. 323±350. Academic Press, Burlington, MA. Guiles, R.D., Altman, J., Kuntz, I.D. & Waskell, L. (1990) Structural studies of cytochrome b5: complete sequence-specific resonance assignments for the trypsin-solubilized microsomal ferrocytochrome b5 obtained from pig and calf. Biochemistry 29, 1276±1289. Guiles, R.D., Basus, V.J., Kuntz, I.D. & Waskell, L. (1992) Sequence specific 1H and 15N resonance assignments for both equilibrium forms of the soluble heme binding domain of rat ferrocytochrome b5. Biochemistry 31, 11365±11375. Turner, D.L. (1995) Determination of haem eletronic structure in His-Met cytochromes c by 13C NMR: the effect of the axial ligands. Eur. J. Biochem. 227, 829±837.
q FEBS 2000 76. Shokhirev, N.V. & Walker, F.A. (1998) Co- and counter-rotation of magnetic axes and axial ligands in low-spin ferriheme systems. J. Am. Chem. Soc. 120, 981±990. 77. Rodionov, M.A. & Blundell, T.L. (1998) Sequence and structure conservation in a protein core. Proteins 33, 358±366. 78. Mortuza, G.B. & Whitford, D. (1997) Mutagenesis of residues 27 and 78 modulates heme orientation in cytochrome b5. FEBS Lett. 412, 610±614. 79. Bertini, I., Luchinat, C. & Rosato, A. (1999) The use of propionate a-proton contact shifts as structural constraints. Inorg. Chim. Acta in press. 80. Hunter, C.L., Lloyd, E., Eltis, L.D., Rafferty, S.P., Lee, H., Smith, M. & Mauk, A.G. (1997) Role of the heme propionates in the interaction of heme with apomyoglobin and apocytochrome b5. Biochemistry 36, 1010±1017. 81. La Mar, G.N., Burns, P.D., Jackson, J.T., Smith, K.M. & Langry, K.C. (1981) Proton magnetic resonance determination of the relative heme orientations in disordered native and reconstituted ferricytochrome b5. Assignment of heme resonances by deuterium labeling. J. Biol. Chem. 256, 6075±6079. 82. Koradi, R., Billeter, M. & WuÈthrich, K. (1996) MOLMOL: a program for display and analysis of macromolecular structure. J. Mol. Graphics 14, 51±55.
S U P P L E M E N TA R Y M AT E R I A L The following material is available from http://www.blackwell-science.com/ejb/ Table S1. Chemical shifts (p.p.m.) of oxidized rabbit microsomal cytochrome b5 (A form). Ê ) used oin structure Table S2. Distance constraints (A calculations. Table S3. Pseudocontact shift constraints used for structure calculations. Table S4. Torsion angle constraints. Table S5. Stereospecific assignment for diastereotopic pairs of methylene protons or isopropyl methyl groups.
