X-ray absorption spectroscopy of the dimeric iron site in azidomethemerythrin from Phascolopsis gouldii.

Abstract

X-ray absorption spectroscopy has been used to study the dimeric iron center in azidomethemerythrin from Phascolopsis gouldii Absorption edge data confirm that the two iron atoms are present as Fe(E) and suggest a hexa-coordination site for each of the iron atoms. The extended x-ray absorption fine structure (EXAFS) analysis provides direct structural evidence of a ,u-oxo bridge between the two iron atoms at an average Fe-O distance of 1. Hemerythrin has been studied by a variety of techniques (1-8) in attempts at elucidating the structure of the active center and understanding the mechanism of reversible oxygenation. Mossbauer, magnetic susceptibility, CD, and optical electronic spectroscopy and x-ray crystallography have provided much detail about the iron environment. In particular, the two irons are antiferromagnetically coupled in oxy-and methemerythrins (7). Cystallographic studies show the coordinating groups to be histidine and carboxylate. Resonance Raman spectroscopy has shown that oxygen is bound as the peroxide (1) and has suggested the presence of OH-, H20, or a ,t-oxo bridge in oxy-and methemerythrin (5).Three crystal structures ofazidomethemerythrins have been determined at the Naval Research Laboratory: monomeric myohemerythrin from Themiste zostericola at 5.5-A resolution (9) with model building and partial refinement to 2.5-A resolution (3), low-resolution structures of octameric hemerythrin from Phascolopsis gouldii (10), and trimeric hemerythrin from Siphonosomafunafuti (unpublished results). The structures of the hydroxylmet and azidomet forms of the octameric hemerythrin from T. dyscrita have been determined and refined to 2.2-A resolution by Stenkamp et aL (11,12) at the University of Washington. Initially, there were substantial disagreements between the two models concerning the Fe-Fe distance and the presence ofa ,u-oxo bridge. More recently, through a series of revisions and refinements at higher resolution, a common model has emerged for the azidomethemerythrin active center. It consists of two hexa-coordinate u-oxo-bridged iron atoms ligated to the protein by five histidyl residues and two bidentate carboxylate bridges.These structures have greatly enhanced our understanding of the hemerythrin active center. Much desired detail is, however, still missing. Electronic information such as the oxidation state is not directly accessible from the crystal structure. Definitive evidence for the presence of a p.-oxo bridge is important, as is verification of the Fe-Fe distance. In addition, structures of the active oxy and deoxy forms (as well as of other met states) have yet to be determined crystallographically. Xray absorption spectroscopy (XAS) has proven useful for investigation of the metal active center in metalloproteins and metalloenzymes (13-15). We have used XAS to examine five derivatives (including the oxy and deoxy) of hemerythrin from P. gouldii and report here the result of our study on the most stable state of the protein, azidomethemerythrin. First, we examine the i...

Binuclear iron complexes in methemerythrin and azidomethemerythrin at 2.0-.ANG. resolution

“…While the difference is not large, it is nominaly significant and can be justified on the basis of the different geometry of Fe(2) in the two complexes. The distances we observe can to be compared with the value of 3.34 (±0.07) Á reported by Hendrickson et al 21 for the Fe-Fe distance in the azidomet complex of myohemerythrin, based on the anomalous scattering differences in the X-ray diffraction data. Values of the Fe-Fe distance derived from EXAFS data are 3.4922 and 3.38 (±0.05) Á,21 these both being measurements of the Fe-Fe distances in azidomethemerythrin from P. gouldii.…”

Anomalous diffraction in crystallographic phase evaluation

“…We had also learned from our experience in determining the structure of crambin from the weak anomalous scattering of Cu Kα x-rays by its intrinsic sulfur atoms that native anomalous scattering can suffice (Hendrickson & Teeter, 1981), and our experience in x-ray absorption spectroscopy of hemerythrin (Hendrickson et al, 1982) indicated that iron proteins might be excellent candidates. Thus, we successfully used lamprey hemoglobin as a first test subject (Hendrickson, 1985; Hendrickson et al, 1988).…”

Resonance Raman study on the active-site structure of a cooperative hemerythrin

“…The protein is typically an octamer with Mr = ~108 000 (Klippenstein, 1980;Klotz et al, 1976;Ward et al, 1975), and each monomer contains a nonheme iron binuclear center which reversibly binds one molecule of oxygen (Boeri & Ghiretti-Magaldi, 1957). The met form of Hr is known to have two high-spin Fe3+ ions with a large antiferromagnetic exchange coupling (Schugar et al, 1972) mediated by an endogenous µbridge, the presence of which was demonstrated by X-ray crystallographic (Stenkamp et al, 1985) and EXAFS studies (Hendrickson et al, 1982).Recently, the structures of the deoxy and oxy forms were solved at the level of 2.0-Á resolution, and the coordination environments around two irons were revealed in detail (Holmes et al, 1991); one iron (Fel) is always hexacoordinated, but the other (Fe2) is penta-or hexacoordinated depending on the two states. Fel and Fe2 are coordinated by three and two histidine residues, respectively, and are joined by the carboxyl side chains of glutamic and aspartic acid residues besides the µbridge.…”

“…The protein is typically an octamer with Mr = ~108 000 (Klippenstein, 1980;Klotz et al, 1976;Ward et al, 1975), and each monomer contains a nonheme iron binuclear center which reversibly binds one molecule of oxygen (Boeri & Ghiretti-Magaldi, 1957). The met form of Hr is known to have two high-spin Fe3+ ions with a large antiferromagnetic exchange coupling (Schugar et al, 1972) mediated by an endogenous µbridge, the presence of which was demonstrated by X-ray crystallographic (Stenkamp et al, 1985) and EXAFS studies (Hendrickson et al, 1982).…”