Single-crystal EPR Study of Novel Azide Complex of Cyanogen Bromide-modified Myoglobin

Hori, Hiroshi; Fujii, M; Shiro, Yoshitsugu; Iizuka, Tetsutarō; Adachi, Shin‐ichi; Morishima, Isao

doi:10.1016/s0021-9258(18)83608-0

Cited by 9 publications

(3 citation statements)

References 14 publications

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“…When azide binds to the sixth coordination site of the heme of metmyoglobin, it acts as a relatively strong-field ligand resulting in a thermal mixture of the high-spin (S ) 5 / 2 ) and low-spin (S ) 1 / 2 ) states at room temperature. Magnetic susceptibility, MCD, and EPR studies of Mb azide all confirm that S ) 1 / 2 is the ground state (Eglinton et al, 1983;Hori et al, 1989;Iizuka, I., & Kotani, 1968;Iizuki, T., & Kotani, 1968). Absorption and MCD studies show the system to be still ∼80% in the low-spin form at room temperature (Smith & Williams, 1968;Vickery et al, 1976).…”

Section: Discussionmentioning

confidence: 89%

CytochromebofromEscherichia coli: Binding of Azide to Cu_B

et al. 1996

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Azide binds to fast cytochrome bo with a stoichiometry of 1:1, the dissociation constant for this reaction being approximately 2 x 10(-5) M. The changes induced in the electronic absorption are very slight and are consistent with heme o remaining hexacoordinate high-spin, an observation confirmed by room temperature MCD spectroscopy in the region 350-2000 nm. X-band EPR spectroscopy of the azide-bound form shows heme o remains coupled to CuB, but that the integer spin signal (g = 3.7) that we have previously reported to be associated with the binuclear center of fast cytochrome bo [Watmough et al. (1993) FEBS Lett. 319, 151-154], is shifted to higher field. The kinetics of azide binding are an order of magnitude faster than those observed for the binding of cyanide. Unlike cyanide, the observed rate constants do not saturate in the range 0.05-25 mM. The value of Kon shows a marked dependence on pH, indicating that the active species is hydrazoic acid. It is argued that these data are consistent with the binding of azide ion as a terminal ligand to CuB yielding a binuclear center in the form FeIII-OH2:: CuBII-N3. The binding of azide in heme-copper oxidases may cause displacement of another nitrogenous ligand from CuB which might explain the absence of electron density associated with histidine-325 in the structure of the Paracoccus denitrificans CCO [Iwata et al. (1995) Nature 376, 660-669]. Formate appears to act as a bidentate ligand to the binuclear center-, blocking not only the binding of azide to CuB but also the binding of cyanide to heme o.

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Section: Discussionmentioning

confidence: 89%

CytochromebofromEscherichia coli: Binding of Azide to Cu_B

et al. 1996

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“…These unusual results allowed us to expect that the exogenous azide ligand could react with BrCN-Mb to form a stable novel complex of ferric Mb. Single-crystal EPR measurements suggested that this complex was not a simple BrCN-MbN3~complex but a novel Mb complex in which the N-cyano group would react with N3~t o form an N-tetrazole-substituted His and that the tetrazole group might coordinate to the heme iron (Hori et al, 1989). This proposed structure of the novel Mb derivative with the N-tetrazole group attached to the distal histidyl imidazole (tetrazole-Mb) was later confirmed by the X-ray structural study (Kamiya et al, 1991).…”

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confidence: 98%

“…On the other hand, we have been interested in facile and site-specific ways to modify the distal His of Mb, that is, chemical modification of the distal His (Shiro & Morishima, 1984;Morishima et al, 1985Morishima et al, , 1989Hori et al, 1989). Although it is difficult to introduce diverse functional groups as site-directed mutagenesis can do by utilizing chemical modification, this method has an advantage in introducing functional groups other than natural amino acid residues very * Correspondence should be addressed to this author, t Present address: Photon Factory, National Laboratory for High Energy Physics, Tsukuba, Ibaraki 305, Japan.…”

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confidence: 99%

Modification of the distal histidyl imidazole in myogloblin to N-tetrazole-substituted imidazole and its effects on the heme environmental structure and ligand binding properties

Adachi

Morishima²

1992

Biochemistry

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The mechanism of N-tetrazole ring formation at the distal histidyl imidazole of sperm whale myoglobin (Mb) has been studied by nitrogen-15 (15N) NMR spectroscopy by utilizing 15N-labeled cyanogen bromide (BrCN) and azide ion (N3-). The 15N-NMR spectrum of BrC15N-modified Mb + N3- afforded two hyperfine-shifted 15N resonances, both of which are identical with the resonance positions of two of the three 15N resonances for BrCN-modified Mb + 15NN2-. This unusual spectral feature is due to the formation of the N-tetrazole ring attached to the distal histidyl imidazole and the scrambling of the labeled nitrogen originated from N3- or BrCN over the tetrazole ring upon coordination to the ferric heme iron. The ferric iron-bound N-tetrazole ring comes off upon reduction to the ferrous state, and the stable CO complex of tetrazole-modified Mb (tetrazole-Mb) is formed. Electronic absorption and 1H-NMR spectra of deoxy and carbonmonoxy forms of tetrazole-Mb are slightly altered from those of native Mb by the modification, while the most significant effect is exerted on the C-O stretching frequency of iron-bound CO. The C-O stretching band for tetrazole-MbCO is observed at 1966 cm-1 in contrast to 1945 cm-1 for native MbCO, suggesting that the geometry of iron-bound CO in tetrazole-Mb is relatively upright which is characteristic of the "open" conformer. This result corresponds to the 15-fold increase of the CO association rate constant by the N-tetrazole modification of the distal His. The oxy form of tetrazole-Mb is readily autoxidized to its ferric state, indicating that hydrogen bonding between the distal His and iron-bound oxygen is essential for stable O2 binding to the heme iron.

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