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 X-ray diffraction studies have produced ABSTRACT a low resolution image and also located the iron atoms of a monomeric hemerythrin from muscles of a sipunculan worm. These results reveal the course of the polypeptide chain and some details of the active center.

Many compounds seem to bind, but derivative crystals are often prone to cracking and poor isomorphism. Nevertheless, five derivatives were found to be good enough at least for lowresolution phasing. X-ray diffraction data were measured by w-scans on a fourcircle diffractometer. CuKa radiation was used. Native data, including the Bijvoet pairs (hkl and hUl), were collected to 2.8 A spacings. Derivative data were taken out to 3A spacings from hkO, Okl, and hOl reflections and to 5.5 A spacings from Bijvoet pairs. Structure factor amplitudes were corrected for the effects of absorption (5), radiation damage (6), and the Lorentz-polarization factor. Native data were placed on an absolute scale by statistical methods (6, 7), and derivative data were scaled to these, making appropriate allowance for the heavy-atom scattering (6). A (SF)2 Patterson map revealed the positions of a single site in the K3UO2F5 derivative. Then .F Fourier syntheses based on phases derived therefrom permitted interpretation of the other derivatives while automatically referring them all to a common origin. The heavy-atom parameters were refined separately for each derivative. Positions of the iron atoms were derived from a |jFhklI - jF-j 112 Patterson map (8) and then refined. Refinement results for the iron atoms and for the heavy-atom derivatives are shown in Table 1. Phase information from the isomorphous replacement and anomalous scattering data was cast in the ABCD formulation (9) to generate combined phase probability distributions. Anomalous dispersion effects were also used to establish the absolute enantiomorph (6). Fourier syntheses of the structure were computed from centroid phases and figure-of-merit weighted structure amplitudes (10, 11). Information from three derivatives was used in computing a first Fourier map. The 490 terms included at 5.5 A resolution had a mean figure-of-merit, mn, of 0.83. A second map based on all five derivatives had mn = 0.89. Finally, the phases were improved by a solvent constraint refinement procedure (manuscript in preparation) to yield a third map with mn = 0.92.

Oxygen transport in certain invertebrate animals is mediated by hemerythrin in erythrocytes of the coelomic fluid. Hemerythrin usually occurs as an octamer of 108,000 molecular weight. It is a non-heme iron protein containing two iron atoms per subunit and it reversibly binds oxygen in the ratio 1 02:2 Fe. Much study has been attended to the structural chemistry of this evolutionary alternative to hemoglobin as an oxygen carrier (1), particularly by Klotz and coworkers, but many details remain obscure. New light can now be shed on hemerythrin structure following the discovery by Klippenstein et al. (2) that the sipunculan worm Themiste (syn. Dendrostomum) pyroides (3) contains a monomeric hemerythrin in its retractor muscles as well as hemerythrin octamers in its erythrocytes-a situation reminiscent of myoglobin and hemoglobin in mammals. Several properties of this myohemerythrin suggest that it bears close structural similarity to the protomers of octameric hemerythrin (2, 4). Myohemerythrin from T. pyroides has been crystallized (4) and the first structural results from studies of these crystals are reported here. These results are mainly at low resolution, but owing to a high helix content and recourse to chemical data, more molecular detail has been gleaned than is ordinarily discernible at low resolution. By way of warning it should be noted that published interpretations of low-resolution density maps have sometimes later been proved incorrect. However, it seems unlikely that such mistakes are repeated here. The quality of this map and the consistency of the model with independent chemical data argue for the basic correctness of the rather detailed molecular interpretation given here. Myohemerythrin is a relatively small protein of 118 aminoacid residues and molecular weight 13,900. This facilitates its crystallographic analysis. In turn, the knowledge of this structure should simplify the analysis of octameric hemerythrins. In any event, further studies on myohemerythrin should be an avenue for gaining a detailed understanding of reversible oxygenation in this fascinating class of proteins.

Interpretation of Fourier maps

The interpretation of even the first Fourier synthesis of myohemerythrin was uncommonly straightforward. Apart from one ambiguity, the bounds of a single contiguous molecule were readily apparent. Most salient among features in the density maps are four rather parallel dense rods, each 30-40 A long, and an especially dense spheroidal mass, 7-10 A in diameter, which they embrace. A more tortuous stretch of density is appended to one side of these features. The ambiguity arose in determining the mode of attachment of this arm to the main body of density. An initial interpretation of this attachment has been abandoned in favor of an alternative which is more reasonable on several grounds. It has

X-ray analys

Crystals of metazide myohemerythrin were grown as previously described (4) and then transferred to a stabilizing medium of 80% saturated ammonium sulfate buffered to pH 6.7. These crystals are in space group P2,2121 and have unit cell dimensions of a = 41.58 A; b = 80.03 A; and c = 37.78 X. Derivatives were prepared by soaking native crystals in stabilizing media containing heavy-atom compounds. 2160

Structure of Myohemerythrin

Proc. Nat. Acad. Sci. USA 72 (1976)

2161

TABIE 1. Parameters from heavy atom refinements Derivative Hg(CN)2

Aa/a 1.73%

Ab/b

0.57%

Ac/c 0.03%

KAu(CN)-

0.50

0.09

0.30

K3UO2F5 K2Pt(CN)4

-0.55 -1.88

-0.05 0.60

-0.26 -0.71

1.03

0.10

-0.34

K1PtCI4

Native anomalous

P

z

B

R

0.475 0.407 0.471 0.497 0.266 0.840 0.444 0.139 0.521 0.579

0.273 0.331 0.290 0.222 0.158 0.176 0.045 0.269 0.630 0.076

18 ,2 10 11 8 16 11 13 9 25 54

0.51

0.206 1.44

0.49

0.070 1.68

0.51 0.47

0.056 1.59 0.226 1.92

0.44

0.127 1.65

0.539 0.479

0.548 0.561

0.323 0.379

q

x

Hgl Aul Au2 U1 Ptl Pt2 Pt3 Ptll Ptl2

0.84 0.62 0.42 0.28 0.47 0.93 0.49 0.42 0.85 0.44

Fel Fe2

0.80 0.92

Hg2

Q

y 0.746 0.807 0.746 0.728 0.451 0.519 0.416 0.773 0.485 0.554

Atom

5 4

0.40

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 improved in successively better-phased maps while that of the alternative model deteriorated. Moreover, the favored model is more compact and has an arm length closer to that expected from the sequence alignment proposed below. A stereo view of the final electron density for the isolated molecule is shown in Fig. 1. This molecular density can be circumscribed by an ellipsoid with axes of 30 X 44 X 28 A along a, b, and C. The electron density distribution of the molecule comprises two entwined parts. One is the dense, rather isolated mass which, for reasons discussed below, must be associated with the iron atoms. The other is a continuous chain of density which courses around this mass through the dense rods and appended arm. By virtue of their straightness, high density, and roughly circular cross sections of 5-6 A diameter, the rods can surely be identified as helical regions of the protein. Stretches of density along the arm and in corners between rods are more crooked and often narrower and less dense. These portions probably correspond to polypeptide segments of more-or-less extended configuration. Fig. 2 imparts a diagrammatic conception of the molecular structure given by this interpretation. The course of the polypeptide chain can be traced by following the locus of highest electron-density points connect-

ing the local maxima of the map. This "ridge" of highest densities has a mean electron density of 0.77 e/A3 along the entire chain. In helical stretches this central density averages 0.85 e/X3 and ranges from 0.72 to 0.94 e/A3. The highest density ridge along the arm ranges from 0.62 to 0.86 e/A3. Average density values both along the arm and through the corners are 0.71 e/.XA. A much higher density of 1.32 r/A3 marks the peak of the iron mass. By contrast, the level of solvent density averages 0.39 e/A3. Each point in the map includes an Fooo contribution of 0.42 e/A3 and has a standard deviation of 0.07 e/A3 as estimated from phase and structure amplitude errors (6, 11). Further insight into the structure can be gained by recourse to chemical evidence about myohemerythrin and to the vast body of information accumulated on the octameric hemerythrin from Golfingia gouldii. Among these data are the aminoacid sequences of T. pyroides myohemerythrin (J. L. Cote, S. E. Ludlam, and G. L. Klippenstein, manuscript in preparation) and of G. gouldii hemerythrin (12). Two other pieces of data provide benchmarks for an alignment of the sequence with electron density features. First, the mercury sites (Table 1) presumably locate the two cysteinyl positions (2). Secondly, the four histidine residues which have been implicated as ligands to the iron atoms (13, 14) can be posited to be at density connections from helical rods to the iron mass.

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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 rubbertubing model of the molecule oriented approximately the same as in Fig. 1.

Lengths of the chain were measured along the contour of separate segments between the benchmarks, ends of rods, and chain termini. Under the assumption of an axial translation of 1.5 A/residue along helices and 3.3 to 3.8 A/residue along lengths of apparently extended chain, particular segments of the sequence were assigned to individual stretches of electron density. Far greater uncertainty must be assessed to these assignments than would be to higher resolution results. However, they are fully consistent with existing data and are helpful in understanding the structure. Thus, the specific assignments are ventured in Table 2. Finally, approximate azimuthal positions of the side chains of helical residues were predicted from helical wheel diagrams (15) oriented according to the benchmarks. Protein conformation

The tertiary folding of myohemerythrin is quite simple. In terms of the standard view shown in Figs. 1 and 2, the chain runs through the N-terminal arm from the molecular bottom, first forth and then back along the left side of the molecule. At the very top, it turns acutely and goes through the first helix (A) to the far end of the molecule, makes a short corner and returns via a second helix (B). Thereupon, the chain moves downward in a U-turn, courses through a third helix (C) and returns through another helix (D). The density at the end of D is rather flattened and weak, indicating a distorted or perhaps even non-helical region. From this point the chain turns sharply upward and to the right into a short carboxyl terminal stub, which in consideration of its density and diameter is probably also helical (E). Further appreciation of the molecular conformation can be gained from Fig. 3. The four principal helices all lie quasi-parallel to b and in cross-section form an approximately rhombic array centered around the iron mass. Interaxial separations of

helices related as the edges of the rhomb are 10-12 A; the AC and BD helix-pairs, related as the diagonals, are 18 A and 15 A apart. Adjacent helices pack together at 100 to 220 away from parallel (Table 2). The resolution of whether or not these angles arise from a "knobs-into-holes" packing (16) must await a more detailed model. Approximately 75% of the residues in this model of myohemerythrin are in helices, which agrees well with estimates from circular dichroism measurements (2, 17). There appears to be no 13-sheet structure. An interesting feature of hemerythrin primary structure is an apparent sequence repeat, albeit at a low level of homology. Residues 18-54 of G. gouldii hemerythrin (12) show 24% homology with residues 67-101 in an alignment which has single residue gaps following 70 and 84. When due allowance is made for five residues inserted into the CD corner of T. pyroides myohemerythrin, this sequence shows 32% homology for the repeat. These segments correspond respectively to major portions of the AB and CD helix-pairs. Inspection of the model discloses that these helix-pairs are approximately related by a local diad passing through the iron mass roughly parallel to b. The correlations among the interhelix angles given in Table 2 are a manifestation of this apparent pseudosymmetry. To test and quantify the pseudosymmetry, least-squares refinements were made seeking the transformation giving the best match of electron density distributions when the CD pair is rotated and translated onto the AB pair. Transformation by a general screw of 171° with 4.7 A translation produced a correlation coefficient of 0.60 between the two density distributions while the match for a pure 2-fold rotation was 0.52. These correlation coefficients are appreciably lower than those from similar comparisons of essentially identical molecules in a- and y-chymotrypsin (0.73-0.77) (18) and in two

Proc. Nat. Acad. Sci. USA 72

Structure of Myohemerythrin

(1975)

(a)N,,D-C

TABIE 2. Strutral features of myohemerythrin Sequence alignment Structural element Residues Amino terminal arm 1-16 A helix 17-37 AB corner 38-39 B helix 40-63 BC corner 64-68 69-87 C helix 88-92 CD corner D helix 93-112 DE corner 113 E helical stub 114-118

2163

(b) N2, A-B

Interhelix angles Helix pair

Angle, 0 4

A-B B-C A-C C-D A-D B-D D-E

158 170 25 163 169 18 80

y

(c) N

(d)D,A

(e)C-B

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 results). Thus, the symmetry is inexact although a significant relationship clearly exists. The local pseudosymmetry in myohemerythrin calls to mind other examples; namely, those in carp muscle calcium-binding protein (19) and in bacterial ferredoxin (20). Dimeric iron center

Studies of M6ssbauer spectra (21-23), magnetic susceptibility measurements (24, 25), circular dichroic spectra (26), and electronic absorption spectra (25-27) have shown that the two iron atoms in hemerythrin must be close together. In particular, the iron atoms in ligand-hemerythrin complexes are antiferromagnetically coupled, probably via a IM-oxo bridge. Thus it was clear at once that the dense spheroidal mass in the Fourier maps must be associated with the iron atoms. That this is so was confirmed by the iron atomic positions (Table 1). The iron-iron distance between these positions is 3.44 A 0.05 A. (The standard deviation here derives from the propagation of errors in lattice constants and in positional parameters as determined by the least-squares refinement. It does not account for possible systematic errors or imperfections in formulation for the least-squares problem.) Assuming iron-oxygen distances of 1.80 A, this distance implies an Fe-O-Fe bridging angle of 1450, which is at the low end of the range for model oxo-bridged dimeric iron complexes (28). Six density connections are made from the protein chain to the iron mass. Four of these, one from each major helix, are about in a plane perpendicular to the local pseudodiad. The other two are from the near end of the molecule, one from the carboxyl stub and another from the BC corner. The positions of these connections are such as to indicate that it is Tyr67, His73, and His106 which are coordinated to Fel, and that His25, His54, and Tyrll4 are coordinated to Fe2. These indications agree with many chemical modification studies which have narrowed the list of potential ligands down to these six residues plus Tyr8 and possibly His77 (13, 14, 29-32). However, they do conflict with one study which reports that Tyr67 is not involved (29). Also, no direct role in iron coordination is seen for Tyr8.

z

FIG. 3. Partial projections of the electron density map of an isolated myohemerythrin molecule. Each section of the density distribution has been contoured and projected onto a common 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 c-axis. An orthogonal view, along a, is given in frames (c), (d), and (e). Each series comprises an entire molecule divided into nonoverlapping segments in the individual frames. Frame (a) shows the lower half of the molecule, including the first part of the N-terminal arm, the D-helix and E-helical stub, C-helix, and part of the iron mass. Frame (b) is of the upper half of the molecule and consists of the N-terminal arm upward from the elbow, the A and B helices, and the upper half of the iron mass. Frame (c) shows the N-terminal arm from the left side of the molecule. Frame (d) is of the middle section of the molecule, 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 B and C and the right half of the iron mass. Boundaries of the frames are x = (0 to 21)a/26, y = (14 to 45)b/48, and z = (-1 to 18)c/24.

The iron center is insulated from the surrounding medium by a cage formed of the four major helices on the sides, the E-helix stub and BC corner at the near end, and the AB and CD corners at the remote end. Aside from the iron ligands, residues predicted to be in proximity to the iron center are generally non-polar. Access to the active center from the left is occluded by the N-terminal arm and from below by a protuberance from the D-helix. However, other chinks between helices may allow limited access. This study does not reveal the binding site for oxygen and anionic ligands, but on steric considerations it is probably in the cavity opposite the apex of tyrosine ligands. Although there is nothing to show the location of the azide ligand, there is evidence concerning its orientation. These crystals are highly dichroic. They appear deep orange in light polarized parallel to b, a rather weak yellow-orange in apolarized light and a pale greenish yellow in c-polarization. The 445 nm. band which is thus polarized has been identified as due to an azide ligand to iron atom charge-transfer transition (26, 27). This implies that the N3 axis lies virtually perpendicular to c and fairly parallel to b.

2164

Biophysics: Hendrickson et al.

Possible octamer interfaces

The interaction between subunits in an octamer is an important feature of hemerythrin structure. The present structure does not bear directly on this matter, of course. However, since there is 46% (54/118) sequence homology between G. gouldii hemerythrin and T. pyroides myohemerythrin and full conservation of putative iron ligands, it seems reasonable to assume that the protomers of octameric hemerythrin are essentially isostructural with myohemerythrin. Given that assumption, molecular surfaces which might possibly be octamer interfaces can be inferred from the correspondence of chemical data with residue positions approximated from this structure. Chemical modification studies on G. gouldii hemerythrin have implicated Cys50 (33), either Tyri8 or Tyr7O (30, 31), one histidine (13), and one or more carboxylates (32) in

subunit interactions. Direct involvement of lysines in subunit binding has been excluded by one report (31), but another study concludes that one or more of the lysines are apparently somehow involved (14). The top surfaces of the A and B helices in a G. gouldii protomer would include Cys5O, His34, Tyr18, exposed hydrophobic residues, and just one peripheral lysine; thus this area qualifies as a probable subunit interface. Substitutions at positions on the AB surface of myohemerythrin may explain its incompetence for association. By contrast, most areas on the right side and bottom seem unlikely as candidates for intersubunit contacts, since many lysine residues and the sites of sequence variability in octamers (34, 35) would be clustered on the outer surfaces of helices C, D, and E. There is no specific evidence implicating the left side of the protomer in subunit interactions, but neither is it disallowed; the N-terminal arm contains no lysines. The diffraction patterns from crystals of G. gouldii hemerythrin indicate the presence of molecular 422 symmetry (36), and T. dyscritum hemerythrin crystallographically expresses at least 2-fold and possibly 4-fold symmetry (37). An octamer of 422 symmetry utilizing surfaces compatible with the chemical data can be constructed by the operations of a 4-fold axis running roughly parallel to c and passing just to the left of the N-terminal arm together with an intersecting diad about parallel to a and passing just above and midway along the A and B helices. The area of surface contact would be quite appreciable. Moreover, such a structure would be 50 A thick axially and 68-78 A in diameter, which is compatible with the proposed crystal packing for G. gouldii hemerythrin. For want of further evidence this octameric structure cannot presently be considered more than an interesting speculation.

Proc. Nat. Acad. Sci. USA 72

(1975)

2. Klippenstein, G. L., VanRiper, D. A. & Oosterom, E. A. (1972) J. Biol. Chem. 247, 5959-5963. 3. Stephen, A. C. & Edmonds, S. J. (1972) The Phyla Sipuncula and Echiura [British Museum (Nat. Hist.), London]. 4. Hendrickson, W. A. & Klippenstein, G. L. (1974) J. Mol. Biol. 87, 147-149. 5. North, A. C. T., Phillips, D. C. & Mathews, F. S. (1968) Acta Crystallogr. Sect. A 24, 351-359. 6. Hendrickson, W. A., Love, W. E. & Karle, J. (1973) J. Mol. Biol. 74, 331-361. 7. Karle, J. & Hauptman, H. (1953) Acta Crystallogr. 6, 473476. 8. Rossmann, M. G. (1961) Acta Crystallogr. 14, 383-388. 9. Hendrickson, W. A. & Lattman, E. E. (1970) Acta Crystallogr. Sect. B 26, 136-143. 10. Blow, D. M. & Crick, F. H. C. (1959) Acta Crystallogr. 12, 794-802. 11. Dickerson, R. E., Kendrew, J. C. & Strandberg, B. E.

(1961) Acta Crystallogr. 14, 1188-1195. 12. Klippenstein, G. L., Holleman, J. W. & Klotz, I. M. (1968) Biochemistry 7, 3868-3878. 13. Fan, C. C. & York, J. L. (1969) Biochem. Biophys. Res. Commun. 36, 365-372. 14. Morrissey, J. A. (1971) Ph.D. Dissertation, Univ. of New Hampshire. 15. Schiffer, M. & Edmundson, A. B. (1967) Biophys. J. 7, 121-135. 16. Crick, F. H. C. (1953) Acta Crystallogr. 6, 689-697. 17. Darnall, D. W., Garbett, K., Klotz, I. M., Aktipis, S. &

Keresztes-Nagy, S. (1969) Arch. Biochem. Biophys. 133,

103-107. 18. Cohen, G. H., Matthews, B. W. & Davies, D. R. (1970) Acta Crystallogr. Sect. B 26, 1062-1069. 19. Kretsinger, R. H. (1972) Nature New Biol. 240, 85-87. 20. Adman, E. T., Sieker, L. C. & Jensen, L. H. (1973). J.

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21. Okamura, M. Y., Klotz, I. M., Johnson, C. E., Winter, M. R. C. & Williams, R. J. P. (1969) Biochemistry 8, 1951-

1958.

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24. 25. 26. 27. 28. 29. 30. 31.

We thank Diane Ward for her drawing of the molecule, Dr. J. Karle for encouragement and support, and Dr. I. M. Klotz for a suggestion. Dr. J. S. Loehr first brought the genus name Themiste to our attention and Dr. M. E. Rice of the Smithsonian Institution confirmed its acceptance as the senior synonym for Dendrostomum. This work was supported in part by the Office of Naval Research and in part by the National Science Foundation.

32.

1. Klotz, I. M. (1971) in Subunits in Biological Systems, eds. Timasheff, S. N. & Fasman, G. D. (Marcel Dekker, New York), pp. 55-103.

37.

33. 34. 35. 36.

38.

& Williams, R. J. P. (1971) Arch. Biochem. Biophys. 142, 574-583. Moss, T. H., Moleski, C. & York, J. L. (1971) Biochemistry 10, 840-842. Dawson, J. W., Gray, H. B., Hoenig, H. E., Rossman, G. R., Schredder, J. M. & Wang, R.-H. (1972) Biochemistry 11, 461-465.Garbett, K., Darnall, D. W., Klotz, I. M. & Williams, R. J. P. (1969) Arch. Biochem. Biophys. 103, 419-434. Keresztes-Nagy, S. & Klotz, I. M. (1965) Biochemistry 5, 919-931. Mabbs, F. E., McLachlan, V. N., McFadden, D. & McPhail, A. T. (1973) J. Chem. Soc. Dalton Trans. 2016-2021. York, J. L. & Fan, C. C. (1971) Biochemistry 10, 1659-1665. Rill, R. L. & Klotz, I. M. (1971) Arch. Biochem. Biophys. 147, 226-241. Fan, C. C. & York, J. L. (1972) Biochem. Biophys. Res. Commun. 47, 472-476. Klippenstein, G. L. (1972) Biochem. Biophys. Res. Commun. 49, 1474-1479. Keresztes-Nagy, S. & Klotz, I. M. (1963) Biochemistry 2, 923-927. Klippenstein, G. L. (1972) Biochemistry 11, 372-380. Ferrell, R. E. & Kitto, G. B. (1971) Biochemistry 10, 2923-2929. North, A. C. T. & Stubbs, G. J. (1974) J. Mol. Biol. 88, 125-131. Loehr, J. S., Meyerhoff, K. N., Sieker, L. C. & Jensen, L. H. (1975) J. Mol. Biol. 91, 521-522. Matthews, B. W. (1966) Acta Crystallogr. 20, 230-239.