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The electronic transitions of the binuclear ferric active site of methemerythrin and oxyhemerythrin (oxyHr) are assigned using variable temperature circular dichroism and polarized single crystal absorbance spectroscopies. These transitions are correlated with crystallographic information to obtain insight into the electronic structure of the oxo-bridged binuclear high spin ferric active site. Ligand field bands are assigned to each iron, and the energy shifts of these bands with variation of exogenous ligand are used to evaluate the ligand field strength of these ligands, particularly the peroxide of oxyHr. Peroxide is found to bind as a very strong field ligand, with both strong u-donor and very weak n-donor character. The exogenous ligand to Fe(II1) charge-transfer spectrum shows that both azide and peroxide have a strong bonding interaction at the active site in addition to the bond to the iron. This is consistent with protonation of the peroxide as suggested from earlier Raman data and with the ligand field strength of peroxide in oxyHr. The intense UV transitions are assigned as oxo-Fe charge transfer and are used to generate a model of the electronic structure of the oxo-bridged iron dimers and in particular the change in the Fe-oxo bond with variation in Fe-0-Fe angle. These results provide insight into the reversible binding of dioxygen by the Hr active site. (1) (a) Okamura, M. Y.; Klotz, I. M. Inorganic Biochemistry; Eichhorn, G. L., Ed.; Elsevier: 1973; pp 320-343. (b) Kurtz, D. M., Jr.; Shriver, D. F.; Klotz, I. M. Coord. Chem. Gray, H. B.; Hoenig, H. E.; Rossman, G. R.; Schredder, J. M.; Wang, R.-H. Biochemistry 1972, 11, 461-465. (c) Okamura, M. y.; Klotz, 1. M.; Johnson, C. E.; Winter, M. R. C.; Williams, R. (7) Stenkamp, R. E.; Sieker, L. C.; Jensen, L. H. J. Am. Chem. SOC. 1984, 106, 618-622. (8) Stenkamp, R. E.; Sieker, L. C.; Jensen, L. H.; McCallum, J. D.; (9) The terminology for met'H20" or met-aquo" Hr is from early work (10) Note. however. a weak C1--Fe(II1) CT transition does, in fact, Sanders-Loehr, J. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 713-716. on the protein" which indicated H20 was bound to the metal site. contribute to the 300-400-nm region (vide infra). Biochem. Biophys. 1969, 135, 419-434. 0 1989 American Chemical Societv (11) Garbett, K.; Darnall, D. W.; Klotz, I. M.; Williams, R. J. P. Arch. , , I -2004

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Chemical and spectroscopic consequences of allosteric interactions for ligand binding to sipunculid (Phascolopsis gouldii) and brachiopod (Lingula reevii) hemerythrins (Hrs) have been investigated. Possible allosteric effectors for homotropic effects in sipunculid Hrs have been examined, but only reduction in ligand affinity is observed without cooperativity. In contrast to sipunculid Hr, L. reevii Hr binds O2 cooperatively in the pH range 7-8 and exhibits a Bohr effect. Spectroscopic comparisons of the sipunculid and brachiopod Hrs show no significant differences in the active site structures; therefore, modulation of oxygen affinity is attributable to effects linking the site to quaternary structural changes in the octamer. Oxygen equilibria can be fit with a conformational model incorporating a minimum of three states, tensed (T), relaxed (R), and an R-T hybrid. Resonance Raman spectra of L. reevii oxyHr show a shift in the peroxo stretching frequency when the pH is lowered from pH 7.7 (predominantly R oxyHr) to pH 6.3 (a mixture of R, T, and R-T hybrid), but P. gouldii Hr does not have a frequency shift under the same conditions. In contrast to hemoglobins, ligand binding to the deoxy and met forms is noncooperative for brachiopod (and sipunculid) Hrs. It is thus suggested that conformational changes in the protein are linked to the oxidation state change that accompanies oxygenation of the coupled binuclear iron site (deoxy [FeIIFeII]----oxy [FeIIIFeIII]). The total allosteric energy expended in oxygenation is about 1.4 kcal/mol, and such a shift is possible in the relaxed-tense conversion with relatively limited constraints of the iron coordination environment via the protein quaternary structure. The mechanism of cooperativity in the binuclear copper oxygen carrier hemocyanin is discussed in light of these results.