Proc. Nat. Acad. Sci. USA Vol. 73, No. 2, pp. 349-351, February 1976

Biochemistry

Structure of the iron complex in methemerythrin (coordination/x-ray diffraction/hemerythrin)

R. E. STENKAMP*, L. C. SIEKER, AND L. H. JENSEN Department of Biological Structure, University of Washington, School of Medicine, Seattle, Wash. 98195

Communicated by Irving M. Klotz, November 13,1975

The coordination of the ligands about the ABSTRACT iron atoms in methemerythrin from Themiste dyscritum has been deduced from a 2.8 A resolution electron density map. The complex can be described in terms of two trigonal antiprisms about the pair of iron atoms in each subunit, the antiprisms having one face in common. Ligands at eight of the nine coordination positions are protein side chains, the ninth presumably being water. Comparison of the electron density map for T. dyscritum methemerythrin with the sequence of Phascolopsis gouldii hemerythrin suggests six aromatic side chain ligands (five histidine and one tyrosine) and two nonaromatic side chain ligands. The latter provide atoms at two of the three vertices of the face shared by the two antiprisms, and these along with the presumed water at the third vertex form bridges between the iron atoms of each pair.

use of anomalous scattering from the Hg and I atoms as was done in the earlier study. There is considerable "noise" in the electron density map, however, in part because the structure is a large one but also because the phases were based on a single derivative only. Nevertheless, there is good correspondence between the electron densities in the four independent subunits for much of the main chain and for many of the side chains, and where questions of interpretation arise for one subunit, they can often be answered by reference to the other subunits. In the 2.8 A map the electron densities at the iron sites were found to be two to three times greater than those of most other peaks in the map, and the Fe atoms in three of the four independent subunits were clearly resolved. Thus the directions of the Fe-Fe vectors within each of the four independent subunits were readily determined, being almost parallel to the crystallographic c axis. From crystal symmetry it follows that the Fe-Fe vectors in all other subunits are likewise almost parallel to the c axis (12). For the purpose of describing the iron complex, we take the line passing through the two Fe atoms in a subunit as vertical. The electron density map shows three side chain ligands coordinated at the vertices of an approximately equilateral triangle, A in Fig. 1, below one of the iron atoms of each pair, and similarly at the vertices of an equivalently oriented triangle above the other iron atom, C in Fig. 1. Two other side chains are visibly fused to the midpoints of each iron complex, presumably providing, at two of the three vertices of triangle B in Fig. 1, bridging ligand atoms between the two Fe atoms. Note that the orientation of triangle B is

Hemerythrin is a protein, found in the erythrocytes of certain marine invertebrates, which functions in oxygen transport. The molecule is an octamer of mass about 107,000 daltons, each subunit containing a pair of iron atoms and binding one 02 molecule (1-3). Despite its name and in contrast to hemoglobin, no heme is present. Since the molecule has properties quite different from those of hemoglobin, yet functions in oxygen transport, it is important to provide a structural basis for understanding its properties. Although chemical and physical studies have revealed many details of the hemerythrin structure (4-10), until recently no three-dimensional model was available which could serve to correlate these studies. The structure of myohemerythrin, which is thought to be similar to the subunit of hemerythrin, has been reported at 5.5 A resolution by Hendrickson et al. (11), and more recently the structures of

hemerythrins from Themiste (Dendrostomum) dyscritum and Phascolopsis (Golfingia) gouldii have been solved at 5 A and 5.5 A resolutions respectively (12, 13). We have now extended the resolution of hemerythrin from T. dyscritum to a nominal value of 2.8 A, and while we were interpreting the electron density map it became apparent that the coordination about the iron pairs in the subunit is quite different from any of the proposed models. Accordingly, we report here the structure of the iron complex as revealed by our present 2.8 A map. Crystals of methemerythrin, space group P4, grow as truncated prisms from solutions of low ionic strength or by dialysis against 35% 2-methyl-2,4-pantanediol. The tetragonal unit cell has dimensions a = b = 86.6 A, c = 80.8 A and contains two octameric molecules. Thus the crystallographic asymmetric unit is composed of four subunits. Intensity data to 2.8 A resolution were collected, as in the 5 A resolution study (12), by a five-step scan technique for crystals of the native protein and one mercury iodide derivative. Phases were determined from the single derivative by *

3C

Present address: Dept. of Molecular Biophysics and Biochemistry, Box 1937 Yale Station, Yale University, New Haven, Conn. 06520.

FIG. 1. Idealized representation of coordination polyhedra about Fe atoms in subunit of T. dyscritum hemerythrin.

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Proc. Nat. Acad. Sci. USA 73 (1976)

Biochemistry: Stenkamp et al.

C2

%-.WI~ ~

~ ~ C

C3

'IO

B2 FIG. 2. Sections from the 2.8 A resolution electron density map. Each frame is a composite of two 12 A square x,y sections separated by a distance of 0.81 A and corresponding approximately to the planes of triangles A, B, and C in Fig. 1. The cross in the middle of each frame is the projected position of the Fe atoms. Although electron density at A-1 and C-1 appears to terminate, additional sections would show connections to the protein main chain.

such that its vertices are staggered with respect to those of A and C. The third vertex of this triangl& is probably occupied by a ligand, presumably water; in any case it does not appear to be a protein side chain. Various groups could probably coordinate at this position, and it suggests itself as the site which may function in oxygen transport. The coordination about the Fe atoms is octahedral and best described in terms of a trigonal antiprism, one face being common to the antiprisms about the two Fe atoms of each pair as shown in Fig. 1. Sections from the 2.8 A electron density map near the planes of triangles A, B, and C in Fig. 1 are shown in Fig. 2 A, B, and C, respectively. The amino-acid sequence for T. dyscritum hemerythrin is known only in part (J. S. Loehr, personal communication), and without a complete sequence it is not possible from the present electron density map to determine with certainty either the identity of the ligand side chains or the sequence numbers of the amino acids involved. Nevertheless, comparison of our map with the sequence of hemerythrin from P. gouldii (14) suggests that there is a high degree of correspondence between the ligands in the two proteins and that they occur in similar if not identical positions in the chain. The amino acid residues in the sequence of P. gouldii hemerythrin corresponding to the ligand positions in Fig. 1 are the following: His 25, A-1; His 54, A-2; Gln 58, B-1; His 73, C-1; His 77, C-2; His 101, C-3; Asp 106, B-2; and Tyr 109, A-S. Although some of the corresponding ligands in T. dyscritum hemerythrin may turn out to differ, we note that the ligand side chains in our map at the vertices of triangles A and C corresponding to histidine and tyrosine in the P. gouldii sequence appear as relatively large, bulky groups for most of the 24 in the four independent subunits. In contrast the eight side chain ligands to B-1 and B-2 are considerably less bulky. We also find a high degree of correspondence between the P. gouldii sequence and bulky side chains in other parts of our map. Chemical studies have implicated both histidine and tyrosine as ligands (1, 4-8), and the appearance, as noted above, of these residues as ligands at the vertices of triangles A and C (Fig. 1) is consistent with these studies. Moreover, the coordination of Tyr at only one of the Fe atoms in a pair is consistent with the observation that when the tyrosine residues are modified by treatment with tetranitromethane only one of the two Fe atoms is lost (5, 6). Mossbauer spectra, on the other hand, are interpreted as indicating similar environments for the Fe atoms in met- and deoxyhemerythrin (15). The possibility of carboxyl ligands has also been recognized (1) although chemical modification of all 18 carboxyl groups

in P. gouldil hemerythrin would seem to rule them out (9). Several of the ligand side chains suggested by our map and the coordination about the Fe atoms differ from the proposal based on the 5.5 A resolution studies of myohemerythrin and hemerythrin from P. gouldli (11, 13, 16). Thus, corresponding to C-1 and C-2 in Fig. 1, the proposed ligands are the side chains of Tyr 67 and His 73, respectively, instead of His 73 and His 77. This is a helical section of the molecule, however, and the positions of ligands at C-1 and C-2 are readily determined relative to other residues in the helix. In our map the last residue in the helix is a large, bulky one in all four subunits and would correspond to Trp 87 in the P. gouldll sequence. Counting back in the helix fixes the ligands at C-1 and C-2 as 73 and 77 relative to 87. No ligands corresponding to B-1 and B-2, Fig. 1, were proposed for myohemerythrin, but at 5.5 A resolution they would not be distinguishable from surrounding electron density. The possibility of substantial differences in the structures of myohemerythrin and the hemerythrins from P. gouldii and T. dyscritum must still be considered, but the remarkable correspondence between our 2.8 A resolution map and the P. gouldii sequence suggests a high degree of similarity between the two hemerythrin structures. We thank Wayne Hendrickson for helpful comments and for copies of manuscripts prior to publication and Joann Loehr who provided hemerythrin from T. dyscritum. This work was supported by Grant AM-3288 from the National Institutes of Health. 1. Klotz, I. M. (1971) Biological Macromolecules, eds. Timasheff, S. N. & Fasman, G. D. (M. Dekker, Inc., New York), Vol. 5, pp. 55-103. 2. Keresztes-Nagy, S. & Klotz, I. M. (1963) Biochemistry 2, 923-927. 3. Klotz, I. M. & Keresztes-Nagy, S. (1963) Biochemistry 2, 445-452. 4. Fan, C. C. & York, J. L. (1969) Biochem. Biophys. Res. Commun. 36,365-372. 5. Rill, R. L. & Klotz, I. M. (1970) Arch. Biochem. Biophys. 136, 507-514. 6. Rill, R. L. & Klotz, I. M. (1971) Arch. Biochem. Biophys. 147, 226-241. 7. York, J. L. & Fan, C. C. (1971) Biochemistry 10, 1659-1665. 8. Fan, C. C. & York, J. L. (1972) Biochem. Blophys. Res. Commun. 47,472-476. 9. Klippenstein, G. L. (1972) Biochem. Blophys. Res. Commun. 49, 1474-1479.

Biochemistry: Stenkamp et al. 10. Dunn, J. B. R., Shriver, D. F. & Klotz, I. M. (1975) Biochemistry 14, 2689-2695. 11. Hendrickson, W. A., Klippenstein, G. L. & Ward, K. B. (1975) Proc. Nat. Acad. Sci. USA 72, 2160-2164. 12. Stenkamp, R E., Sieker, L. C., Jensen, L. H. & Loehr, J. S. (1975) J. Mol. Biol., in press. 13. Ward, K. B., Hendrickson, W. A. & Klippenstein, G. L. (1975)

Proc. Nat. Acad. Sci. USA 73 (1976)

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Nature 257, 818-821. 14. Klippenstein, G. L., Holleman, J. W. & Klotz, I. M. (1968)

Biochemistry 7,3868-3878. 15. Okamura, M. Y., Klotz, L. M., Johnson, C. E., Winter, M. R. C. & Williams, R. J. P. (1969) Biochemistry 8,1951-1958. 16. Hendrickson, W. A. & Ward, K. B. (1975) Biochem. Blophys. Res. Commun. 66,1349-1356.

Structure of the iron complex in methemerythrin.

The coordination of the ligands about the iron atoms in methemerythrin from Themiste dyscritum has been deduced from a 2.8 A resolution electron densi...
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