Structure of alkaline metmyoglobin-xenon complex

J. Mol. Biol. (1969) 45, 297-303

Structure of Alkaline Metmyoglobin-Xenon BENNO

Complex

P. SCHOENBORN

Biology Department, Brookhaven National Laboratory Upton, New York 11973, U.S.A. (Received 10 October 1968, and in revised form 16 June 1969) Structural changes of alkaline metmyoglobin compared to neutral metmyoglobin have been determined by a three-dimensional X-ray diffraction analysis. Alkaline metmyoglobin crystals proved, however, to be somewhat unstable under X-irradiation. Exposure of crystals to xenon stabilized them thus permitting the necessary extended X-ray exposures. Structural changes were determined by inspection of difference electron density maps with subsequent refinement by a difference least-squares method. The results show two xenon binding sites in addition to a number of structural rearrangements.

1. Introduction Crystallographic studies of various myoglobin derivatives have demonstrated that cyanide and hydroxide myoglobin are significantly different from met-, deoxy- and oxymyoglobin (Nobbs, 1966; Watson & Chance, 1966; Watson & Nobbs, 1968). The latter are nearly identical, apart from changes at the sixth iron co-ordination position. These preliminary X-ray crystallographic investigations also showed that the cyanide complex, a low spin compound, and the hydroxide, a mixed spin compound, apparently share a number of features not found in the other myoglobin derivatives. During early attempts to obtain X-ray diffraction data from alkaline metmyoglobin crystals it became apparent that these crystals are somewhat unstable under X-irradiation (see below). Exposure of these crystals to xenon stabilized them however and permitted the extended exposures needed for three-dimensional data collection. During investigations of the functional changes induced in myoglobin by the presence of xenon, Chance & Settle (personal communication) showed that the binding of carbon monoxide and the hydroxide formation were altered (Pig. 1) while the oxygen and carbon dioxide binding functions were unaffected. This stabilizing effect of xenon on the hydroxide myoglobin crystals therefore increased interest in the elucidation of the alkaline metmyoglobin-xenon structure.

2. Methods Sperm whale metmyoglobin was prepared by the method of Parrish & Kendrew (1956) and crystallized from an 80% ammonium sulfate solution at pH 6.9. After completion of crystallization the pH was adjusted to 9.4 with NaOH. Initial cracking of crystals was observed, but considerable recrystallization took place within a 3-week period during which the pH gradually decreased to 9.1. All crystallographic work was carried out with crystals at pH 9.1; it was shown spectroscopically that complete conversion to the hydroxide form is achieved at pH 8.9. Crystals prepared in this manner proved to be somewhat

unstable under X-irradiation

with diffraction intensities decreasing rapidly after exposures 297

298

B.

P. SCHOENBORN

pH 6.0

100365

/ OD.

7

pH 7.7

I

23 rnMNo OH

86 lb. Xenon on

Almin

k-

630 to 700 mp FIG+. 1. Double beam spectrogram of metmyoglobins caused by NaOH and by Xe (courtesy of B. Chance).

transition

to the alkaline

met form

as

of a few hours. Exposure to xenon stabilized these crystals and permitted three-dimensional data collection. Crystals were mounted in quartz capillaries and equilibrated with xenon at 2.6 atmospheres. The diffraction data were collected to 2.8 A resolution, using the conventional precession technique with unfiltered copper K radiation from a sealed X-ray tube. The crystals did not remain sufficiently long in the hydroxide form (20 hr) to permit the use of a nickel filter. Intensities were measured on a Joyce-Loebel recording microdensitometer, corrected for Lorentz and polarization factors and then sealed to the native metmyoglobin data. A three-dimensional difference Fourier map was then calculated using 2666 independent reflections and the standard metmyoglobin phases kindly provided by Dr J. C. Kendrew. These same phases were used for the structural calculations that led to the recently published myoglobin co-ordinates of Watson (1968) and were originally determined by Kendrew and coworkers (1962) who noted negative peaks at the gold and mercury sites. Subsequently, the atomic position, but not the phases, were refined by a block-diagonal least-squares program developed by C. I. Branden with constraints introduced by Watson (personal communication). At present, however, it is unclear whether the metmyoglobin phases and the atomic positions could be improved by Branden’s blockdiagonal least-squares method using the relaxed restraints suggested by Rollett & Koenig (personal communication). It is also possible, of course, that these observed negative regions are due to structural changes inherent in the particular heavy-atom derivatives used. In order to determine more exactly the changes suggested by the difference map, a modified full matrix least-squares technique was used. The Busing-Levy (1961) leastsquares program as adapted by LaPlace 8~ Ibers (1965) for group refinement was modified structure-factor differences. The squared quantities to enable refinement on “phased” minimized in this process, given in equation (I), are explained by Fig. 2.

structure factor with COFor additional or ejected atoms: F, is the normal calculated ordinates determined from the difference map. For groups : FG is the difference between the structure factors calculated for the altered group (F,,*) and the structure factors (Fc,x) calculated from the original atomic positions in the native metmyoglobin structure. This is achieved either by subtracting the native atomic structure factors for the groups in question before actual least-squares calculations, or by introducing a group twice in the refinement program, first as an invariant with fixed negative contribution and then as a variant with a positive contribution. In the group refinement, individual atomic structure factors are calculated, but the group is kept intact by refining only the “origin” and orientation of the groups. A particular group system can be arranged so as to allow only e.g. rotation around a single bond. Such an approach one degree of rotational freedom,

m

,.:. .:

;

,. r.-, 1? . :; 8. _

*

,::: ‘, ‘\ . -’

ALKALINE

METMYOGLOBIN

299

FIG. 2. Fn observed amplitude of derivative, e.g. Mb Xe (OH); Fp native protein amplitude with phase ar; AF = F, - P,; (AF,) = FD - F,; calculated from AF and (ar - orn); an, phase of derivative calculated initially from the native phase and the primary features depicted in the difference map, for each least-squares cycle Q, is recalculated on the basis of the previous cycles refined parameters; c+, phase of native protein. permits the use of a full matrix least-squares refinement in cases where only a small number of parameters change. For the particular application described, the program allowed refinement of 20 individual atoms and 16 groups with up to 40 atoms each. The maximum number of permissible variables was 168.

3. Results (a) Difference electron density map An inspection of the difference electron density map calculated with data to 2.8 A reveals two primary features with equal peak heights of 0.9 eAm3. The use of a set of only approximate phases in a difference Fourier synthesis results in reduced peak heights. Luzatti (1953) showed that this reduction depends on the proportion of centric to non-centric reflections and on the magnitude of the structural changes. In this case where the change in electron density is relatively small, a 5Oyb reduction in peak height can be expected. These two peaks of nearly equal height would then correspond to scattering centers with about 45 electrons each. All other peaks in the map are considerably smaller and are located mainly in the region of the E helix (Plate I), the G helix with the GH corner (Plate I), and the space between molecules. One of the primary peaks is at the xenon site found in met and reduced myoglobin (Schoenborn, Watson & Kendrew, 1965; Schoenborn & Nobbs, 1966), a site which is buried in the interior of the molecule and located roughly equidistant from the hemelinked histidine and a pyrrole group of the heme. Peak 2 lies between the AB and GH corner, roughly halfway between the histidines GHl and B5. The difference map shows a number of other features in this area indicating shifts of the two histidines; other minor peaks indicate some rearrangement in the GH corner and the G helix. Distance calculations from peak 2 show that no covalently bonded group could reside there without disturbing the two histidines as found in metmyoglobin. One of the major secondary features, -0.4 eAe3 (peak 3 in Plate III), corresponds to the loss of a negatively charged group on the surface of metmyoglobin. This peak is at the same site as one observed in both azide myoglobin (Stryer, Kendrew 6 Watson, 1964) and deoxymyoglobin (Nobbs, Watson & Kendrew, 1966) and has been interpreted as being due to the loss of a negative ion, possibly sulfate, from NS of histidine E7 and Gl’i. The only other significant feature located within the molecule is a sequence of positive and negative peaks along the E helix starting at about E9 and progressing to the EF corner. In the space between molecules, a number of features are observed depicting changes 20

B.

300

P. SCHOENBORN TABLE

1

Groups used in the least-squares re$nement, with molecular reorientation given as rotations along speciJied bonds Alteration

Group Histidine

Helical

B5 Cl FG3 GHI Cl4 Cl7 segment ElO-El8

+20”f8” (Ca-Cfi); - 15”&8” (Cj3-Cy) +25’=f8” (&-C/3) - 2o”* 6” (cc&g) +62”&10’ (Cc&/J); +50”&20” (Cfi-Cy) uncertain; change 15Of 15’ uncertain; change lo”* 10” twist, + 10’13” along rtxis of helix kink, + 7’* 3” raises end of helix in y

in ion and water structures as well as rearrangement of groups previously polar but now, at pH 9.1, non-polar. Some of these clearly indicate molecular rearrangements, while others are suggestive of some changes that cannot readily be interpreted in terms of structural changes. This seems to be especially true for groups on the molecular surface that are also ill defined in the native metmyoglobin structure. The clearly indicated structural changes are listed in Table 1 and were subsequently refined by the least-squares method previously described. The other features of the map indicating structural perturbations whose exact nature could not be clearly determined are listed in Table_2. TABLE 2

Croups with minor alterations

as indicated

Possible change

Group Glutrtmic acid A2 Glutamic aoid A4 Tryptophan A5 Lysine Al4 Glutamic acid A16 Lysine E20 Histidine EF4 Histidine G14 Veline G15 Leuoine G16 Histidine Cl7 Serine Cl8

in difference map

ion on NH could be missing ion between NE of tryptophan A5 and carboxyl group of A4 shift of side chain ion on carboxyl with H bond to lysine E20 broken; with side chain shift movement of ring to +z, +y indicated ring and peptide shift to -z peptide shift to -z peptide shift to --z peptide and ring to --z peptide shift to --z

(b) Rejkement For the least-squares refinement, as described above, only those groups (Table 1) were used which showed clear changes in the difference map and which were clearly defined in the original metmyoglobin Fourier. These groups were refined with rotational freedom about a defined axis centered on the origin of a given group (e.g. C/3 in histidine), since it had been noted that significant rotations only took place about permissible axes (e.g. about Cu-C/? in histidine). A refinement sequence permitting

ALKALINE

METMYOGLOBIN

301

positional changes in the group origin was also calculated. In addition, the occupancy and positions were refined for peaks 1, 2 and 3 (Table 3). All these calculations were carried out with unit weighted difference data and with constant temperature factors. In order to test this refinement procedure, phenylalanine (H14) (which showed no difference peaks at all) was also introduced, but showed only rotation of 1” about the Ca-C/3 bond and 0.1 A displacement in the group origin; both numbers were well within TABLE

Aaadkd

3

features with rejked fmction4d co49dinates ,A+3

z Peak 1 Xe 1 Pet&2202 Peak 3 Sulfate

0.175 (0.177)1 0.401 O-369

2

Y 0.868 (0.864) 0.400 0.066

0.163 (0.168) 0.622 0.272

t The fractional co-ordinates for peak 1 as found metmyoglobin are given in parentheses.

from Fourier

Weight in electrons from least squares

0.89 0.89 -0.34

in the two-dimensional

+49

+26 -21 analysis

in xenon

the expected error. The results of this analysis are summarized in Table 1. Only histidine GHl moved from its anchor point (Ccc), all other groups remained at their origin within an error of 0.2 A. The group origin of GHl was lowered by 1-O f O-2 d. The difference map indicates not only a lowering for GHl but also for GH2, GH3 and G17. Further refinement of this region was, however, not attempted since this corner was ill defined in the original Fourier map and phase improvement for the native structure is deemed necessary before changes can be reliably postulated. In the refinement of the E helical regions from El0 to EM, groups of different lengths were used, all of which showed a general tendency to a clockwise twist in the helix. The final refinement was carried out with the entire helical segment from El0 to El8 intact. This showed a twist of 10 f 3” starting probably in the E9 region.

4. Discussion The only changes observed in the heme group were a small possible shift of CNPR (see labeling of heme atoms in Schoenborn, Watson & Kendrew, 1965) and shift of histidine FtG3 which must result in a break of the hydrogen bond between NE of FG3 and an oxygen of the propionic side chain. The absence of this hydrogen bond may explain why the xenon derivative of the hydroxide myoglobin forms more stable crystals. The loss of this hydrogen bond on increasing the pH would loosen the heme group and, therefore, decrease the stability of the heme within the protein. The presence of xenon (peak 1) located between the hemelinked histidine and a pyrrole group of the heme would, however, prevent the heme from moving (steric restraints). The structure surrounding this site shows no perturbation. The co-ordinates found for this peak 1 differ by less than 0.2 d from the xenon position found in the two-dimensional analysis of xenon metmyoglobin. This xenon atom with 90% occupancy, occupies therefore the same site with an identical environment. Peak 2 with a height of O-9eAm3 has been tentatively identified as a xenon atom

B.

302

P. SCHOENBORN

with 50% occupancy. This peak lies at the site of the gold atom in the gold chloride derivative, a site which shows a deep negative hole in the original myoglobin electron density map and as peaks of 0.32 and 0.53 eAe3 in the deoxymyoglobin and azide myoglobin difference analyses. These latter two peaks were presumed to be due to errors in phase determination. In the present case, however, the peak is considerably larger and presents a composite made up of the above-mentioned error peak and a “true” component which refined well in the least-squares procedure. Further, the peak lies at a point in the structure where two histidines (GHl and B5) change their positions due to the loss of hydrogen atoms (bond) at this high pH of 9.1. The new histidine positions determined by the group least-squares analysis would again permit a London bonded xenon atom (Table 4). Xenon 2 lies again between a non-polar area TABLET Approach distances to xenon 2, distances < 5.5 A Group Histidine

B5

Histidine

GHl

Atom COr C& NC2 Cc1 N& N CCf W CY C& NC2 Cc, N& 0 g?J

Distance 5.4 4.8 3.9 3.5 4.4 4.9 5.0

5.0

0 :;

4.1 4.1 3.7 3.6 4.0 5.3 5-l 4.3 4.2 4.6 5.0

Valine Al5

N CU (3

4.4 4.4 6.4

Leucine G16 Arginine Cl9

2 1 0 :;

5.2 5.3 3.9 4.3 5.0

: NH, a

5.5 5-4 4.5 5.4

Veline Al 1 Lysine

Al4

and an area that is partially polar. Xenon 2 has 31 neighbors in van der Waals’ contact. This suggests that this complex is partly stabilized by dipole-induced dipole moments but mainly by London interactions. The magnitude of the London interaction can again be calculated from the electronic dispersion interaction to yield an interaction energy of the order of 10 kcal. The bonding configuration at the second xenon site does not exist in the metmyoglobin-xenon complex where the

ALKALINE

METMYOGLOBIN

303

above-mentioned histidines are linked by hydrogen bonds to other groups. It should be noted that this second xenon site corresponds approximately to the xenon position found in hemoglobin (Schoenborn, 1965), although no explanation for this coincidence is apparent. Since no change was observed at the sixth iron co-ordination position, which is occupied by a water molecule in metmyoglobin (Nobbs, Watson & Kendrew, 1966), it must be concluded that the heme ligand is either an OH ion or a water molecule in the alkaline metmyoglobin derivative. The distal histidine shows no positional change itself but the loss of a group with 24 electrons bound to N6 of E7 and G17 is observed (peak 3). This change could be caused by the loss of the hydrogen atom on G17 or E7 due to the high pH or more likely could be caused by an OH ion at the sixth coordination position of the iron. No obvious link could be found between the observed twist in the E helix and functional changes in the molecule ; it is much more likely that these perturbations are entirely due to changes in the ionic surrounding. It has been suggested by Nobbs (personal communication) that these changes could be direct evidence of the onset of alkaline denaturation. All the observed definite changes are connected with imidazole groups of histidine residues, groups which lose a hydrogen at this pH. It should be noted that all the histidines which moved in the alkaline metmyoglobin-xenon complex as compared to metmyoglobin are those that can be alkylated by 0.2 &I-bromoacetate (Gurd & Hugli, 1968 Abstr. Am. Meeting Amer. Chem. BOG.)while those that did not move could not be alkylated. Hi&dine has also relatively dense structural features, where even small shifts produce significant differences in structure amplitudes and are therefore easily observed. There are, however, a number of features observable, associated particularly with lysine, glutamic acid and aspartic acid that might be significant but cannot be uniquely separated from the noise level. This work was supported in part by U.S. Public Health Service Grant NB03625 to R. M. Feather&one, University of California Medical Center, San Francisco and supported in part by the U.S. Atomic Energy Commission. I thank Drs J. C. Kendrew and H. C. Watson for their advice and Mrs J. Knapp and Mr K. Grist for their technical assistance. REFERENCES Busing, W. P. & Levy, H. A. (1961). In Computing Metho& and the Phase ProbZem, p. 146. Oxford : Oxford University Press. Kendrew, J. C., Watson, H. C., Strandberg, B. E., Dickerson, R. E., Phillips, D. C. & Shore, V. C. (1962). Nature, 190, 666. LaPIaca, S. J. & Ibers, J. A. (1965). Acta Cry& 18, 511. Luzzati, V. (1953). Acta Cry&. 6, 142. Nobbs, C. L. (1966). In Hemes and Hemoproteilzs, ed. by B. Chance, R. Estabrook & T. Yonetani, p. 143. New York: Academic Press. Nobbs, C. L., Watson, H. C. & Kendrew, J. C. (1966). Nature, 209, 339. Parrish, R. G. & Kendrew, J. C. (1956). Proc. Roy. Sot. A, 238, 305. Schoenborn, B. P. (1965). Nature, 208, 760. Schoenborn, B. P. & Nobbs, C. L. (1966). Mol. PharmucoZ. 2, 491. Schoenborn, B. P., Watson, H. C. & Kendrew, J. C. (1965). Nature, 207, 28. Stryer, L., Kendrew, J. C. & Watson, H. C. (1964). J. Mol. Btil. 8, 96. Watson, H. C. (1969). In Progress in LYtereochemistry, vol. 4, in the press. Watson, H. C. & Chance, B. (1966). In Hemee and Hewproteins, ed. by B. Chance, R. Estabrook & T. Yonetani, p. 149. New York: Academic Press. Watson, H. C. & Nobbs, C. L. (1968). Biochemie des Sauerstoffs, Proceedings of 19th Mosbath Colloquium, Berlin: Springer Verlag, p. 37.