Proc. Nat. Acad. Sci. USA Vol. 72, No. 6, pp. 2160-2164, June 1975

Tertiary Structure of Myohemerythrin at Low Resolution (hemerythrin/oxygen transport/sipunculan worm/protein structure/x-ray diffraction) WAYNE A. HENDRICKSON*, GERALD L. KLIPPENSTEINt, AND KEITH B. WARD* *Laboratory for the Structure of Matter, Naval Research Laboratory, Washington, D.C. 20375; and t Department of Biochemistry, University of New Hampshire, Durham, N.H. 03824 Communicated by I. M. Klotz, March 17, 1975

ABSTRACT X-ray diffraction studies have produced Many compounds seem to bind, but derivative crystals are a low resolution image and also located the iron atoms of a often prone to cracking and poor isomorphism. Nevertheless, monomeric hemerythrin from muscles of a sipunculan worm. These results reveal the course of the polypeptide five derivatives were found to be good enough at least for low- chain and some details of the active center. resolution phasing. X-ray diffraction data were measured by w-scans on a four- Oxygen transport in certain invertebrate animals is mediated circle diffractometer. CuKa radiation was used. Native data, by hemerythrin in erythrocytes of the coelomic fluid. He- including the Bijvoet pairs (hkl and hUl), were collected to merythrin usually occurs as an octamer of 108,000 molecular 2.8 A spacings. Derivative data were taken out to 3A spacings weight. It is a non-heme iron protein containing two iron from hkO, Okl, and hOl reflections and to 5.5 A spacings from atoms per subunit and it reversibly binds oxygen in the ratio Bijvoet pairs. Structure factor amplitudes were corrected for 1 02:2 Fe. Much study has been attended to the structural the effects of absorption (5), radiation damage (6), and the chemistry of this evolutionary alternative to hemoglobin as Lorentz-polarization factor. Native data were placed on an an oxygen carrier (1), particularly by Klotz and coworkers, absolute scale by statistical methods (6, 7), and derivative but many details remain obscure. New light can now be shed data were scaled to these, making appropriate allowance for on hemerythrin structure following the discovery by Klippen- the heavy-atom scattering (6). stein et al. (2) that the sipunculan worm Themiste (syn. Den- A (SF)2 Patterson map revealed the positions of a single drostomum) pyroides (3) contains a monomeric hemerythrin in site in the K3UO2F5 derivative. Then .F Fourier syntheses its retractor muscles as well as hemerythrin octamers in its based on phases derived therefrom permitted interpretation erythrocytes-a situation reminiscent of myoglobin and of the other derivatives while automatically referring them hemoglobin in mammals. Several properties of this myo- all to a common origin. The heavy-atom parameters were hemerythrin suggest that it bears close structural similarity refined separately for each derivative. Positions of the iron to the protomers of octameric hemerythrin (2, 4). atoms were derived from a |jFhklI - jF-j112 Patterson map Myohemerythrin from T. pyroides has been crystallized (4) (8) and then refined. Refinement results for the iron atoms and the first structural results from studies of these crystals and for the heavy-atom derivatives are shown in Table 1. are reported here. These results are mainly at low resolution, Phase information from the isomorphous replacement and but owing to a high helix content and recourse to chemical anomalous scattering data was cast in the ABCD formulation data, more molecular detail has been gleaned than is ordi- (9) to generate combined phase probability distributions. narily discernible at low resolution. By way of warning it Anomalous dispersion effects were also used to establish the should be noted that published interpretations of low-resolu- absolute enantiomorph (6). Fourier syntheses of the structure tion density maps have sometimes later been proved incorrect. were computed from centroid phases and figure-of-merit However, it seems unlikely that such mistakes are repeated weighted structure amplitudes (10, 11). Information from here. The quality of this map and the consistency of the three derivatives was used in computing a first Fourier map. model with independent chemical data argue for the basic The 490 terms included at 5.5 A resolution had a mean correctness of the rather detailed molecular interpretation figure-of-merit, mn, of 0.83. A second map based on all five given here. derivatives had mn = 0.89. Finally, the phases were improved Myohemerythrin is a relatively small protein of 118 amino- by a solvent constraint refinement procedure (manuscript acid residues and molecular weight 13,900. This facilitates in preparation) to yield a third map with mn = 0.92. its crystallographic analysis. In turn, the knowledge of this structure should simplify the analysis of octameric hemeryth- Interpretation of Fourier maps rins. In any event, further studies on myohemerythrin should The interpretation of even the first Fourier synthesis of be an avenue for gaining a detailed understanding of reversible myohemerythrin was uncommonly straightforward. Apart oxygenation in this fascinating class of proteins. from one ambiguity, the bounds of a single contiguous mole- cule were readily apparent. Most salient among features in X-ray analys the density maps are four rather parallel dense rods, each Crystals of metazide myohemerythrin were grown as pre- 30-40 A long, and an especially dense spheroidal mass, 7-10 A viously described (4) and then transferred to a stabilizing in diameter, which they embrace. A more tortuous stretch medium of 80% saturated ammonium sulfate buffered to of density is appended to one side of these features. The pH 6.7. These crystals are in space group P2,2121 and have ambiguity arose in determining the mode of attachment of unit cell dimensions of a = 41.58 A; b = 80.03 A; and c = this arm to the main body of density. An initial interpretation 37.78 X. Derivatives were prepared by soaking native crystals of this attachment has been abandoned in favor of an alter- in stabilizing media containing heavy-atom compounds. native which is more reasonable on several grounds. It has 2160 Downloaded by guest on October 2, 2021 Proc. Nat. Acad. Sci. USA 72 (1976) Structure of Myohemerythrin 2161 TABIE 1. Parameters from heavy atom refinements

Derivative Aa/a Ab/b Ac/c Atom q x y z B R Q P Hg(CN)2 1.73% 0.57% 0.03% Hgl 0.84 0.475 0.746 0.273 18 ,2 0.51 0.206 1.44 Hg2 0.62 0.407 0.807 0.331 10 KAu(CN)- 0.50 0.09 0.30 Aul 0.42 0.471 0.746 0.290 11 0.49 0.070 1.68 Au2 0.28 0.497 0.728 0.222 8 K3UO2F5 -0.55 -0.05 -0.26 U1 0.47 0.266 0.451 0.158 16 0.51 0.056 1.59 K2Pt(CN)4 -1.88 0.60 -0.71 Ptl 0.93 0.840 0.519 0.176 11 0.47 0.226 1.92 Pt2 0.49 0.444 0.416 0.045 13 Pt3 0.42 0.139 0.773 0.269 9 K1PtCI4 1.03 0.10 -0.34 Ptll 0.85 0.521 0.485 0.630 25 0.44 0.127 1.65 Ptl2 0.44 0.579 0.554 0.076 54 Native anomalous Fel 0.80 0.539 0.548 0.323 5 0.40 Fe2 0.92 0.479 0.561 0.379 4

Structure factor contributions from the heavy atoms were calculated as fH = Zqif(s) exp (- Bis2) exp 27ri(h xi + ky1 + lzi) where s = sin O/A. Scattering factors, f(s), were taken asfo + Af' from tabulations for neutral atoms. The adjustable parameters are q, the fractional occupancy; x, y, and z, the atomic positions in fractions of a cell edge; and B, the isotropic "temperature" factor. The sum is over all sites in the unit cell. For centric reflections, observed amplitudes were taken as AF = FH - Fp, where FH is the structure amplitude for the heavy-atom derivative and Fp is that for the native protein. Probable sign-reversal reflections (6) were excluded from the refinements. For general reflections, observed amplitudes were estimated by Matthews' coefficient (38) formed with a relative weighting for Bijvoet differences chosen such as to equate the averages of these coefficients with averages of AF in the centric zones. These refinements were based on the 668 centric reflections with spacings greater than 3 A and the 300 general reflections to 5.5 A. The number of sign-reversal exclusions ranged from 17 to 153. The refinement of iron atom positions was based on the structure factor contributions from anomalous scattering in the native protein. Observed structure amplitudes were taken as IFk1 - Fai-i/2 and, accordingly, scattering factors were assumed to be f(s) = Af" = 3.45e, independent of s. These observations systematically underestimate the true anomalous scattering amplitude by about a factor cos ' where ip is the phase difference between the anomalous scattering vector and the total "real atom" structure factor. However, the exclusion of weak Bijvoet differences tends to eliminate reflections for which cos 4, is small. Thus, only reflections for which Fhkk - FhkI exceeded one sigma for the difference were included in the refinement. A total of 1504 of the 2627 general reflections at 2.8 A resolution qualified. Nonetheless the effect of cos 4, is not fully compensated so occupancies are somewhat underestimated here. In all cases, the R-value cited is R = ZjFo - FJ1/2Fo where Fo and F, are observed and calculated magnitudes of structure factors against which the refinement was made. Sums are taken over all data included in the refinement. The ratio Q = ((AF)2)/(Fp2) is a measure of the scattering power of the heavy atoms-relative to that of the protein in an isomorphous derivative. These averages are over all centric data at 3 A resolution. The ratio, P = (Jffl)/(C2)V/2, of the average calculated heavy-atom structure amplitude to the rms lack-of-closure error (10) is a measure of the phasing power of a derivative. These averages include all the 5.5 A data.

fewer false joins or weak connections, and its connectivity ing the local maxima of the map. This "ridge" of highest improved in successively better-phased maps while that of densities has a mean electron density of 0.77 e/A3 along the the alternative model deteriorated. Moreover, the favored entire chain. In helical stretches this central density averages model is more compact and has an arm length closer to that 0.85 e/X3 and ranges from 0.72 to 0.94 e/A3. The highest expected from the sequence alignment proposed below. A density ridge along the arm ranges from 0.62 to 0.86 e/A3. stereo view of the final electron density for the isolated Average density values both along the arm and through the molecule is shown in Fig. 1. This molecular density can be corners are 0.71 e/.XA. A much higher density of 1.32 r/A3 circumscribed by an ellipsoid with axes of 30 X 44 X 28 A marks the peak of the iron mass. By contrast, the level of along a, b, and C. solvent density averages 0.39 e/A3. Each point in the map The electron density distribution of the molecule comprises includes an Fooo contribution of 0.42 e/A3 and has a standard two entwined parts. One is the dense, rather isolated mass deviation of 0.07 e/A3 as estimated from phase and structure which, for reasons discussed below, must be associated with amplitude errors (6, 11). the iron atoms. The other is a continuous chain of density Further insight into the structure can be gained by recourse which courses around this mass through the dense rods and to chemical evidence about myohemerythrin and to the vast appended arm. By virtue of their straightness, high density, body of information accumulated on the octameric hemeryth- and roughly circular cross sections of 5-6 A diameter, the rin from Golfingia gouldii. Among these data are the amino- rods can surely be identified as helical regions of the protein. acid sequences of T. pyroides myohemerythrin (J. L. Cote, Stretches of density along the arm and in corners between S. E. Ludlam, and G. L. Klippenstein, manuscript in prepara- rods are more crooked and often narrower and less dense. tion) and of G. gouldii hemerythrin (12). Two other pieces of These portions probably correspond to polypeptide segments data provide benchmarks for an alignment of the sequence of more-or-less extended configuration. Fig. 2 imparts a with electron density features. First, the mercury sites (Table diagrammatic conception of the molecular structure given 1) presumably locate the two cysteinyl positions (2). Secondly, by this interpretation. the four histidine residues which have been implicated as The course of the polypeptide chain can be traced by ligands to the iron atoms (13, 14) can be posited to be at following the locus of highest electron-density points connect- density connections from helical rods to the iron mass. Downloaded by guest on October 2, 2021 2162 Biophysics: Hendrickson et al. Proc. Nat. Acad. Sci. USA 72 (1976)

FIG. 1. Stereoplot of the electron density distribution in an isolated myohemerythrin molecule. The first contour level is at 0.53 e/X3 (-2a above the solvent level) and higher contours are drawn at intervals of 0.22 e/X3 (ha3o). The molecule is here viewed with a directed horizontally from left to right, b running vertically from page bottom (molecular near end) to page top (molecular far end), and c passing from below the page (molecular bottom) to above the page (molecular top). The figure was computer drawn with a program from Dr. B. C. Wishner. FIG. 2. An artist's conception of the molecular structure of myohemerythrin. The figure was drawn by Diane Ward from a rubber- tubing model of the molecule oriented approximately the same as in Fig. 1.

Lengths of the chain were measured along the contour of helices related as the edges of the rhomb are 10-12 A; the separate segments between the benchmarks, ends of rods, and AC and BD helix-pairs, related as the diagonals, are 18 A chain termini. Under the assumption of an axial translation of and 15 A apart. Adjacent helices pack together at 100 to 1.5 A/residue along helices and 3.3 to 3.8 A/residue along 220 away from parallel (Table 2). The resolution of whether lengths of apparently extended chain, particular segments of or not these angles arise from a "knobs-into-holes" packing the sequence were assigned to individual stretches of electron (16) must await a more detailed model. density. Far greater uncertainty must be assessed to these Approximately 75% of the residues in this model of myo- assignments than would be to higher resolution results. How- hemerythrin are in helices, which agrees well with estimates ever, they are fully consistent with existing data and are from circular dichroism measurements (2, 17). There appears helpful in understanding the structure. Thus, the specific to be no 13-sheet structure. assignments are ventured in Table 2. Finally, approximate An interesting feature of hemerythrin primary structure is azimuthal positions of the side chains of helical residues were an apparent sequence repeat, albeit at a low level of homology. predicted from helical wheel diagrams (15) oriented according Residues 18-54 of G. gouldii hemerythrin (12) show 24% to the benchmarks. homology with residues 67-101 in an alignment which has single residue gaps following 70 and 84. When due allowance Protein conformation is made for five residues inserted into the CD corner of The tertiary folding of myohemerythrin is quite simple. In T. pyroides myohemerythrin, this sequence shows 32% terms of the standard view shown in Figs. 1 and 2, the chain homology for the repeat. These segments correspond respec- runs through the N-terminal arm from the molecular bottom, tively to major portions of the AB and CD helix-pairs. first forth and then back along the left side of the molecule. Inspection of the model discloses that these helix-pairs are At the very top, it turns acutely and goes through the first approximately related by a local diad passing through the helix (A) to the far end of the molecule, makes a short corner iron mass roughly parallel to b. The correlations among the and returns via a second helix (B). Thereupon, the chain interhelix angles given in Table 2 are a manifestation of this moves downward in a U-turn, courses through a third helix apparent pseudosymmetry. (C) and returns through another helix (D). The density at To test and quantify the pseudosymmetry, least-squares the end of D is rather flattened and weak, indicating a dis- refinements were made seeking the transformation giving the torted or perhaps even non-helical region. From this point best match of electron density distributions when the CD the chain turns sharply upward and to the right into a short pair is rotated and translated onto the AB pair. Transforma- carboxyl terminal stub, which in consideration of its density tion by a general screw of 171° with 4.7 A translation produced and diameter is probably also helical (E). Further appreciation a correlation coefficient of 0.60 between the two density of the molecular conformation can be gained from Fig. 3. distributions while the match for a pure 2-fold rotation was The four principal helices all lie quasi-parallel to b and 0.52. These correlation coefficients are appreciably lower than in cross-section form an approximately rhombic array those from similar comparisons of essentially identical mole- centered around the iron mass. Interaxial separations of cules in a- and y-chymotrypsin (0.73-0.77) (18) and in two Downloaded by guest on October 2, 2021 Proc. Nat. Acad. Sci. USA 72 (1975) Structure of Myohemerythrin 2163 TABIE 2. Strutral features of myohemerythrin (a)N,,D-C (b) N2, A-B Sequence alignment Interhelix angles Structural element Residues Helix pair Angle, 0 Amino terminal 4 arm 1-16 A-B 158 A helix 17-37 B-C 170 AB corner 38-39 A-C 25 B helix 40-63 C-D 163 y BC corner 64-68 A-D 169 C helix 69-87 B-D 18 (c) N (d)D,A (e)C-B CD corner 88-92 D-E 80 D helix 93-112 DE corner 113 E helical stub 114-118

Assignments of residue positions are estimated as no better than :±:2. Residue positions here refer to the sequence of T. pyroides myohemerythrin. Corresponding positions r in the G. gouldii hemerythrin are related as rHr = rMyoHr for rMyoHr < 90 and as rir = rMyoar -5 for rMyoHr > 95.

crystal forms of bloodworm hemoglobin (0.75, unpublished z results). Thus, the symmetry is inexact although a significant FIG. 3. Partial projections of the electron density map of an relationship clearly exists. The local pseudosymmetry in isolated myohemerythrin molecule. Each section of the density myohemerythrin calls to mind other examples; namely, distribution has been contoured and projected onto a common those in carp muscle calcium-binding protein (19) and in plane. Contour lines are drawn at intervals of 0.18 e/A3 beginning at 0.51 e/1&. The projections in frames (a) and (b) are down the bacterial ferredoxin (20). c-axis. An orthogonal view, along a, is given in frames (c), (d), Dimeric iron center and (e). Each series comprises an entire molecule divided into nonoverlapping segments in the individual frames. Frame (a) Studies of M6ssbauer spectra (21-23), magnetic susceptibility shows the lower half of the molecule, including the first part of measurements (24, 25), circular dichroic spectra (26), and the N-terminal arm, the D-helix and E-helical stub, C-helix, electronic absorption spectra (25-27) have shown that the and part of the iron mass. Frame (b) is of the upper half of the two iron atoms in hemerythrin must be close together. In molecule and consists of the N-terminal arm upward from the particular, the iron atoms in ligand-hemerythrin complexes elbow, the A and B helices, and the upper half of the iron mass. are antiferromagnetically coupled, probably via a IM-oxo Frame (c) shows the N-terminal arm from the left side of the bridge. Thus it was clear at once that the dense spheroidal molecule. Frame (d) is of the middle section of the molecule, mass in the Fourier maps must be associated with the iron showing helices A, D, and E and the left side of the iron mass. Frame (e) shows the right side of the molecule, including helices atoms. That this is so was confirmed by the iron atomic B and C and the right half of the iron mass. Boundaries of the positions (Table 1). frames are x = (0 to 21)a/26, y = (14 to 45)b/48, and z = (-1 The iron-iron distance between these positions is 3.44 A to 18)c/24. 0.05 A. (The standard deviation here derives from the prop- agation of errors in lattice constants and in positional The iron center is insulated from the surrounding medium parameters as determined by the least-squares refinement. by a cage formed of the four major helices on the sides, the It does not account for possible systematic errors or imperfec- E-helix stub and BC corner at the near end, and the AB and tions in formulation for the least-squares problem.) Assuming CD corners at the remote end. Aside from the iron ligands, iron-oxygen distances of 1.80 A, this distance implies an residues predicted to be in proximity to the iron center are Fe-O-Fe bridging angle of 1450, which is at the low end of generally non-polar. Access to the active center from the left the range for model oxo-bridged dimeric iron complexes (28). is occluded by the N-terminal arm and from below by a pro- Six density connections are made from the protein chain tuberance from the D-helix. However, other chinks between to the iron mass. Four of these, one from each major helix, helices may allow limited access. This study does not reveal are about in a plane perpendicular to the local pseudodiad. the binding site for oxygen and anionic ligands, but on steric The other two are from the near end of the molecule, one from considerations it is probably in the cavity opposite the apex the carboxyl stub and another from the BC corner. The of tyrosine ligands. positions of these connections are such as to indicate that it Although there is nothing to show the location of the azide is Tyr67, His73, and His106 which are coordinated to Fel, ligand, there is evidence concerning its orientation. These and that His25, His54, and Tyrll4 are coordinated to Fe2. crystals are highly dichroic. They appear deep orange in light These indications agree with many chemical modification polarized parallel to b, a rather weak yellow-orange in a- studies which have narrowed the list of potential ligands polarized light and a pale greenish yellow in c-polarization. down to these six residues plus Tyr8 and possibly His77 (13, The 445 nm. band which is thus polarized has been identified 14, 29-32). However, they do conflict with one study which as due to an azide ligand to iron atom charge-transfer transi- reports that Tyr67 is not involved (29). Also, no direct role tion (26, 27). This implies that the N3 axis lies virtually in iron coordination is seen for Tyr8. perpendicular to c and fairly parallel to b. Downloaded by guest on October 2, 2021 2164 Biophysics: Hendrickson et al. Proc. Nat. Acad. Sci. USA 72 (1975) Possible octamer interfaces 2. Klippenstein, G. L., VanRiper, D. A. & Oosterom, E. A. (1972) J. Biol. Chem. 247, 5959-5963. The interaction between subunits in an octamer is an impor- 3. Stephen, A. C. & Edmonds, S. J. (1972) The Phyla Sipuncula tant feature of hemerythrin structure. The present structure and Echiura [British Museum (Nat. Hist.), London]. does not bear directly on this matter, of course. However, 4. Hendrickson, W. A. & Klippenstein, G. L. (1974) J. Mol. since there is (54/118) sequence homology between Biol. 87, 147-149. 46% 5. North, A. C. T., Phillips, D. C. & Mathews, F. S. (1968) G. gouldii hemerythrin and T. pyroides myohemerythrin and Acta Crystallogr. Sect. A 24, 351-359. full conservation of putative iron ligands, it seems reasonable 6. Hendrickson, W. A., Love, W. E. & Karle, J. (1973) J. to assume that the protomers of octameric hemerythrin are Mol. Biol. 74, 331-361. essentially isostructural with myohemerythrin. Given that 7. Karle, J. & Hauptman, H. (1953) Acta Crystallogr. 6, 473- 476. assumption, molecular surfaces which might possibly be 8. Rossmann, M. G. (1961) Acta Crystallogr. 14, 383-388. octamer interfaces can be inferred from the correspondence 9. Hendrickson, W. A. & Lattman, E. E. (1970) Acta Crystal- of chemical data with residue positions approximated from logr. Sect. B 26, 136-143. this structure. 10. Blow, D. M. & Crick, F. H. C. (1959) Acta Crystallogr. 12, 794-802. Chemical modification studies on G. gouldii hemerythrin 11. Dickerson, R. E., Kendrew, J. C. & Strandberg, B. E. have implicated Cys50 (33), either Tyri8 or Tyr7O (30, 31), (1961) Acta Crystallogr. 14, 1188-1195. one histidine (13), and one or more carboxylates (32) in 12. Klippenstein, G. L., Holleman, J. W. & Klotz, I. M. (1968) subunit interactions. Direct involvement of lysines in subunit Biochemistry 7, 3868-3878. binding has been excluded by one report (31), but another 13. Fan, C. C. & York, J. L. (1969) Biochem. Biophys. Res. Commun. 36, 365-372. study concludes that one or more of the lysines are apparently 14. Morrissey, J. A. (1971) Ph.D. Dissertation, Univ. of New somehow involved (14). The top surfaces of the A and B Hampshire. helices in a G. gouldii protomer would include Cys5O, His34, 15. Schiffer, M. & Edmundson, A. B. (1967) Biophys. J. 7, Tyr18, exposed hydrophobic residues, and just one peripheral 121-135. lysine; thus this area qualifies as a probable subunit interface. 16. Crick, F. H. C. (1953) Acta Crystallogr. 6, 689-697. 17. Darnall, D. W., Garbett, K., Klotz, I. M., Aktipis, S. & Substitutions at positions on the AB surface of myohemeryth- Keresztes-Nagy, S. (1969) Arch. Biochem. Biophys. 133, rin may explain its incompetence for association. By contrast, 103-107. most areas on the right side and bottom seem unlikely as 18. Cohen, G. H., Matthews, B. W. & Davies, D. R. (1970) candidates for intersubunit contacts, since many lysine Acta Crystallogr. Sect. B 26, 1062-1069. 19. Kretsinger, R. H. (1972) Nature New Biol. 240, 85-87. residues and the sites of sequence variability in octamers 20. Adman, E. T., Sieker, L. C. & Jensen, L. H. (1973). J. (34, 35) would be clustered on the outer surfaces of helices C, Biol. Chem. 248, 3987-3996. D, and E. There is no specific evidence implicating the left 21. Okamura, M. Y., Klotz, I. M., Johnson, C. E., Winter, side of the protomer in subunit interactions, but neither is M. R. C. & Williams, R. J. P. (1969) Biochemistry 8, 1951- no 1958. it disallowed; the N-terminal arm contains lysines. 22. York, J. L. & Bearden, A. J. (1970) Biochemistry 9, 4549- The diffraction patterns from crystals of G. gouldii he- 4554. merythrin indicate the presence of molecular 422 symmetry 23. Garbett, K., Johnson, C. E., Klotz, I. M., Okamura, M. Y. (36), and T. dyscritum hemerythrin crystallographically & Williams, R. J. P. (1971) Arch. Biochem. Biophys. 142, expresses at least 2-fold and possibly 4-fold symmetry (37). 574-583. 24. Moss, T. H., Moleski, C. & York, J. L. (1971) Biochemistry An octamer of 422 symmetry utilizing surfaces compatible 10, 840-842. with the chemical data can be constructed by the operations 25. Dawson, J. W., Gray, H. B., Hoenig, H. E., Rossman, of a 4-fold axis running roughly parallel to c and passing G. R., Schredder, J. M. & Wang, R.-H. (1972) Biochemistry just to the left of the N-terminal arm together with an 11, 461-465.- 26. Garbett, K., Darnall, D. W., Klotz, I. M. & Williams, intersecting diad about parallel to a and passing just above R. J. P. (1969) Arch. Biochem. Biophys. 103, 419-434. and midway along the A and B helices. The area of surface 27. Keresztes-Nagy, S. & Klotz, I. M. (1965) Biochemistry 5, contact would be quite appreciable. Moreover, such a struc- 919-931. ture would be 50 A thick axially and 68-78 A in diameter, 28. Mabbs, F. E., McLachlan, V. N., McFadden, D. & Mc- which is compatible with the proposed crystal packing for Phail, A. T. (1973) J. Chem. Soc. Dalton Trans. 2016-2021. 29. York, J. L. & Fan, C. C. (1971) Biochemistry 10, 1659-1665. G. gouldii hemerythrin. For want of further evidence this 30. Rill, R. L. & Klotz, I. M. (1971) Arch. Biochem. Biophys. octameric structure cannot presently be considered more than 147, 226-241. an interesting speculation. 31. Fan, C. C. & York, J. L. (1972) Biochem. Biophys. Res. Commun. 47, 472-476. 32. Klippenstein, G. L. (1972) Biochem. Biophys. Res. Commun. We thank Diane Ward for her drawing of the molecule, Dr. J. 49, 1474-1479. Karle for encouragement and support, and Dr. I. M. Klotz for a 33. Keresztes-Nagy, S. & Klotz, I. M. (1963) Biochemistry 2, suggestion. Dr. J. S. Loehr first brought the genus name Themiste 923-927. to our attention and Dr. M. E. Rice of the Smithsonian Institution 34. Klippenstein, G. L. (1972) Biochemistry 11, 372-380. confirmed its acceptance as the senior synonym for Dendrosto- 35. Ferrell, R. E. & Kitto, G. B. (1971) Biochemistry 10, mum. This work was supported in part by the Office of Naval 2923-2929. Research and in part by the National Science Foundation. 36. North, A. C. T. & Stubbs, G. J. (1974) J. Mol. Biol. 88, 125-131. 1. Klotz, I. M. (1971) in Subunits in Biological Systems, eds. 37. Loehr, J. S., Meyerhoff, K. N., Sieker, L. C. & Jensen, L. H. Timasheff, S. N. & Fasman, G. D. (Marcel Dekker, New (1975) J. Mol. Biol. 91, 521-522. York), pp. 55-103. 38. Matthews, B. W. (1966) Acta Crystallogr. 20, 230-239. Downloaded by guest on October 2, 2021