Low temperature optical spectroscopy of cobalt-substituted hemocyanin from Carcinus maenas

Abstract

In this work we report the optical absorption spectra of three cobalt-substituted derivatives of hemocyanin (Hc) from Carcinus maenas, in the temperature range 300-20 K. The derivatives studied are the mononuclear (Co 2 +)-Hc with a single cobalt ion in the "CUA" site, the binuclear (Co 2 +)2-Hc and the binuclear mixed metal (Co 2 +-Cu 1 +)-Hc. At low temperature three main bands are clearly resolved; the temperature dependence of their zeroth, first and second moments sheds light on the stereodynamic properties in the surroundings of the chromophore. Within the limits of the reported analysis, in the binuclear derivatives the motions coupled to the chromophore appear to be "essentially harmonic" in the whole temperature range investigated; moreover the data are consistent with the presence of an exogenous ligand strongly bound to the two metal ions. For the mononuclear derivative an "essentially harmonic" behavior is evident only up to 200 K where the data are consistent with the presence of an exogenous ligand much less strongly bound, while at higher temperatures the behavior of the spectra indicates the onset of very large inharmonic contributions to motions, that plausibly involve the above exogenous ligand and, quite likely, the entire active site.Abbreviations: Hc, Hemocyanin; Mo, zeroth moment; Mr, first moment, M2, second moment; (Co2-)2-Hc, blnuclear blcobalt hemocyanin derivative; (Co2+)-Hc, mononuclear monocobalt hemocyanin derivative; (Co 2 +-Cu 1 +)-Hc, binuclear mixed metals hemocyanin derivative; LFT, ligand field theory; CT, charge transfer; EPR, electronic paramagnetic resonance; XANES, X-ray absorption near edge structure

An x-ray absorption near edge structure spectroscopy study of metal coordination in Co(II)-substituted Carcinus maenas hemocyanin

“…Even if from the XANES simulations we cannot exclude the presence of dioxygen bound end-on in cisor trans-mode to the two Co atoms (in this way each Co would be four-coordinated), the cobaltous oxidation state rules out the presence of an oxygenated site. Recent investigations on the temperature dependence of optical spectra of Co(II)-Hc (Vitrano et al, 1993), are consistent with the presence of an exogenous ligand strongly bound to the two metal ions of the dinuclear derivative. Independent structural data giving the Co-Co distance in the dinuclear and mononuclear derivatives could confirm this indication.…”

Cobalt(II) Substituted Derivatives ofCarcinus maenasHemocyanin: Magnetic Characterization, Magnetooptic, and Kinetic Studies Regarding the Geometry of the Active Site

“…The MCD spectra in the 1.4−2.2 μm -1 region of Co II 2 −Hc, Co II 2 −N 3 −Hc, and Co II −Hc at room temperature as well as the Co II 2 −Hc MCD spectrum at 4.2 K are shown in Figure A−C (upper panels) and Figure , respectively; the corresponding energies of the band maxima are presented in Table . In addition, the optical absorption spectra are displayed (Figures , lower panels) to allow a comparison with the absorption maxima previously discussed in the literature. , In Figure A−C) the near-infrared MCD spectra are displayed.

5 (A−C) Optical absorption (upper) and MCD spectra (lower) of (A) Co II 2 −Hc, (B) Co II 2 −N 3 −Hc, and (C) Co II −Hc at room temperature (0.1 M Tris/HCl buffer, pH 8).

6 MCD spectra of Co II 2 −Hc at room temperature (A) and at 4.2 K (B).

7 (A−C) Near-infrared MCD spectra of (A) 1.56 mM Co II 2 −Hc, (B) 1.66 mM Co II 2 −N 3 −Hc, and (C) 1.54 mM Co II −Hc at room temperature (0.1 M Tris/HCl buffer, pH 8).

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“…maenas ) hemocyanin (Hc) containing both a mononuclear and a dinuclear metal center. X-ray absorption near edge structure spectroscopy (XANES) and low-temperature optical spectroscopic studies of the mononuclear and dinuclear derivatives revealed a four-coordinate metal binding site for each metal ion, showing tetrahedral (Co II 2 −Hc) and distorted tetrahedral (Co II −Hc, Cu I −Co II −Hc) geometry, respectively. Earlier studies on Co(II)-substituted L .…”

Low temperature optical absorption spectroscopy: an approach to the study of stereodynamic properties of hemeproteins

“…It is well known that the optical absorption spectra of metalloproteins depend markedly on temperature (Knowles et al 1975;San Biagio et al 1977;Dooley et al 1979;Solomon et al 1980;Browett and Stillman 1984;Allendorf et al 1985;Cordone et al 1986;Leone et al 1987;Di Iorio et al 1991;Vitrano et al 1993); as an example we shown in Fig. 1 the Sorer band of Asian elephant MbCO at various temperatures in the range 300-20 K. Three main effects are observed on lowering the temperature: i) halfwidth narrowing, ii) peak frequency shift, iii) integrated intensity variations.…”

“…This approach has been exploited by our group in recent years (Di Pace et al 1992;Cupane et al 1993 a, b) and is briefly reviewed in this paper. In the "Theoretical Section" we describe the basic theory of the linear coupling between an intense isolated electronic transition and the vibrations of the nearby matrix.…”