The physiologically active forms of the nonheme-iron, oxygen-transport protein hemerythrin have been studied by x-ray crystallographic techniques. At 3.9-A resolution, a difference electron-density map between the deoxy form and met form (methemerythrin) of the protein suggests only small differences in the binuclear iron complexes. The coordination of the iron atoms appears to be the same in both the deoxy and met forms, one iron of the complexes being pentacoordinate, the other iron being hexacoordinate. The iron atoms appear to be somewhat farther apart in the deoxy form. A 2.2-A resolution study of oxyhemerythrin shows that dioxygen binds to one iron atom-the pentacoordinate one in the met form of the protein, the same binding site found for azide in azidomethemerythrin.The initial objective of the crystallographic structure determinations of hemerythrin was to structurally characterize this nonheme-iron, oxygen-transport protein to provide a basis for understanding the chemical differences between its derivatives. The early studies focused on the structure of the oxidized form, or methemerythrins (1, 2), primarily because they are more stable than are the physiologically more interesting deoxy and oxy forms. The active center is a binuclear iron complex bound to the protein by amino acid side chains. In azidomethemerythrin, the iron atoms are both hexacoordinate ( Fig. 1 Left). In the met form [formerly identified as aquomet (3) or hydroxomet (4)], one of the iron atoms, designated Fe2, is pentacoordinate ( Fig. 1 (11), and x-ray absorption spectroscopy (12) indicate that the metal complex in oxyhemerythrin is similar to that in azidomethemerythrin, where the two Fe(III) atoms are bridged by a A-oxygen atom, and the exogenous ligand binds only to one Fe atom. The x-ray crystallographic results reported here support this description for oxyhemerythrin and, in addition, suggest some rearrangement in the binuclear iron complex upon conversion from deoxy to oxy. The 2.2-A resolution crystallographic investigations provide important structural information bearing on the functional properties of the protein and limiting the mechanisms to be considered in explaining the biological action of the molecule. EXPERIMENTALThe sipunculid T. dyscrita was obtained from the Oregon Institute of Marine Biology (Charleston, OR). Hemerythrin was extracted and crystallized from the coelomic fluid as described (13, 14). The crystals were dissolved in 0.2 M Tris sulfate (pH 8.0) and concentrated to -30 mg of protein per ml by ultrafiltration (XM-50 filter; Amicon). The hemerythrin solution was depleted of oxygen by anaerobic dialysis against nitrogen-purged buffer. A 4-fold molar excess (per hemerythrin monomer) of solid sodium dithionite was added to the dialysis buffer. After 25 hr the protein solution displayed the pale yellow color characteristic of reduced hemerythrin. Crystals of deoxyhemerythrin were obtained by slow dialysis of the concentrated solution against 0.02 M Tris sulfate (pH 7.2) under anaerobic condition...

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