Electron paramagnetic resonance crystallography of ¹⁷ O-enriched oxycobaltomyoglobin: Stereoelectronic structure of the cobalt dioxygen system

Abstract

An electron paramagnetic resonance crystallographic study was made on oxycobaltomyoglobin with the dioxygen ligand enriched to 19.1% in 170. There are two srectroscopically distinct cobalt dioxygen species. The less abundant species, II (40%), has nonequivalent oxygen atoms with su- Although the direction cosines for this molecule cannot be precisely determined, the projection of its 0-0 axis ar proximately bisects N2-Fe-N3 and is parallel to the imidazo e ring of His-F8. Increase of temperature changes g, C-A, and OA values, with the largest effect seen with OA. This temperature dependence indicates averaging of the two bond structures which are stabilized at 77 K. The bonding of dioxygen to heme proteins has long been a topic of interest, extensive research activity, and debate. Various techniques and approaches have been brought to bear on the problem of seeking an accurate geometric and electronic description of the metal-dioxygen bond. Because of high electron density near the heme and ease of autoxidation of the ferrous ion, x-ray crystallography has yet to yield a definitive structure of oxymyoglobin or oxyhemoglobin. Recent development of low-temperature x-ray diffraction techniques in several laboratories may overcome these difficulties. Model oxyheme complexes have been synthesized and found by x-ray crystallography to have an Fe-O-O bond angle of t120' (1). One helpful development was the preparation of the Co analogs of hemoglobin (CoHb) and myoglobin (c'Mb) (2); these bind dioxygen reversibly. In the case of COHb the binding of dioxygen is cooperative (2-4). We have performed electron paramagnetic resonance (EPR) crystallography on C°Mb and CoMbO2 and have reported the g and COA tensors (5). At -195°C, two oxy species having the same g but different C6A tensors were observed (5), with one 0-0 axis turned approximately 90°with respect to the other in the crystal. By assuming the principal axes of the g tensor to be coincident with the molecular axes of the dioxygen ligand, the results were inThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- MgO, ZnO, and KCI matrices (7,8). However, a preliminary report on the x-ray structure of CoMbO2 has appeared (9). The data were taken at -16'C and a Co-O-O angle of near 1200 is reported as contrast to the possible ir-bond structure (5). The discrepancy in the CoO-O bond angle must be resolved. Finally, the central purpose of this work was to determine all the hyperfine tensors and relevant eigenvectors in order to arrive at the detailed stereoelectronic structure of C°MbO2. MATERIALS AND METHODSCoMb was prepared and crystallized as described (5) with the addition of a purification step of adsorption onto a CM-cellulose column at pH 6.6 in 0.01 M phosphate and desorption with 0.1 M phosphate buffer at pH 7.5. There was approximately a 30% loss of non-native material at this step. The protein was crystallized over a pH range of 5.98-6.76 and an (NH4)2SO4 concentration ran...

Trapping and spectroscopic characterization of an FeIII-superoxo intermediate from a nonheme mononuclear iron-containing enzyme

“…The 57 Fe hyperfine interaction (A 0 ∕g n β n ¼ −21.4 T) compares well with that of the mononuclear Fe III site (−21.2 T) of protocatechuate 3,4 dioxygenase from Brevibacterium fuscum (27), and the isomer shift (δ ¼ 0.50 mm∕s) falls squarely into the range of high-spin Fe III (25). The 17 O hyperfine interaction of the superoxide radical is large (A17 O ¼ 180 MHz) as in superoxide adsorbed onto various surfaces (33,34) and oxycobaltomyoglobin (35).…”

Probing Hydrogen Bonding to Bound Dioxygen in Synthetic Models for Heme Proteins: The Importance of Precise Geometry

“…173.0, 172.7, 159.4, 157.4, 151.5, 150.1, 149.3, 149.2, 148.1, 147.9, 136.8, 135.8, 131.1, 130.8, 130.3, 130.1, 129.5, 129.2, 128.3, 125.5, 124.0, 122.1, 120.0, 118.6, 116.0, 114.0, 111.2, 109.7, 105.8, 105.4, 104.9, 93.7, 77.3, 67.9, 67.5, 66.8, 60.1, 59.5, 46.8, 30.9, 29.7, 29.5, 29.0, 26.3, 25.5, 23.4, 14.2, 13.6 Zn (1427.98): C 68.13, H 5.86, N 6.87;found: C 68.13, H 5.97, N 6.58. (Tetraethyl 4,4',4'',4'''-{[10-(2-(1H-imidazol-5-ylethynyl)-5-{[6-(1H-[1,3-15 N 2 ]imidazol-1-yl)hexyl]oxy}phenyl)porphyrin-5,15-diyl-kN 21 ,kN 22 , kN = 173.0, 172.7, 159.3, 157.4, 150.1, 149.3, 149.2, 148.1, 147.9, 136.9, 131.1, 130.8, 130.7, 130.3, 130.1, 129.5, 129.2, 123.9, 122.1, 120.0, 118.6, 115.9, 114.0, 111.2, 109.7, 105.8, 105.4, 104.9, 93.7, 77.3, 67.9, 67.5, 66.8, 60.1, 59.5, 46.8, 46.7, 31.9, 30.9, 29.7, 29.6, 29.5, 29.3, 29.1, 29.0, 26.3, 25.5, 24.0, 23.4, 22.7, 14.2, 13. …”

“…Yield 40 mg (74 %); m.p. 102-104 8C; (Tetraethyl 4,4',4'',(4)(5)(6)(7)(8)(9)(10)hexyl]oxy}phenyl)porphyrin-5,15-diyl-kN 21 ,kN 22 ,kN 23 ,…”

“…24,H 5.78,N 7.84;found: C 67.41,H 5.95,N 7.70. (Tetraethyl 4,4',4'',[4][5][6][7][8][9][10]-5-{ [6-(1H-imidazol-1-yl)hexyl]oxy}phenyl)porphyrin-5,15-diyl-kN 21 ,kN 22 ,kN 23 ,kN 24 ]bis[benzene-2,1,3-triylbisA C H T U N G T R E N N U N G (oxy)]}tetrabutanoato(2À))zinc (4-Zn): The title compound was prepared from 18-Zn (50 mg, 0.04 mmol) according to the general procedure using iodinated precursor 21. Yield 40 mg (74 %); m.p.…”

Direct Evidence for a Hydrogen Bond to Bound Dioxygen in a Myoglobin/Hemoglobin Model System and in Cobalt Myoglobin by Pulse‐EPR Spectroscopy

“…Although the hyperfine tensor is not entirely axial, and the bulk electron spin population is not localized at one atom but distributed between the two oxygen nuclei, we assumed the spin density to be centered at one point in space. [13] A distance of (2.3 AE 0.2) between this spin-density center and the NH proton of the distal benzimidazole was obtained by using the pointdipole approximation with a dipolar contribution T = 6.5 MHz.…”