Geometric and electronic structure of oxyhemocyanin: spectral and chemical correlations to met apo, half met, met, and dimer active sites.

Abstract

The chemical and spectral properties of a series of hemocyanin derivatives were systematically compared to provide insight into the geometric and electronic structure of the oxyhemocyanin active site. The binuclear copper site is characterized as two tetragonal Cu(II) Oxygen binds as peroxide (1) to the active site in oxyhemocyanin (2) in the ratio of 1 02:2 Cu and it is generally agreed that the two coppers are in the +2 oxidation state, although a Cu(I)Cu(III) active site has recently been proposed (3). The lack of an electron paramagnetic resonance (EPR) signal for oxyhemocyanin, then, requires the existence of antiferromagnetic coupling or a metal-metal bond between the cupric ions. Magnetic susceptibility studies (4) failed to detect any deviation from Curie behavior, placing only a lower limit of 550 cm-1 on any antiferromagnetic etchange interaction. The optical spectrum also exhibits unique spectral features. The dominant absorption at 345 nm has been variously assigned as a simultaneous pair excitation (SPE) (5) or a ligand-to-metal charge transfer transition (LMCT) with the ligand suggested to be S-(cysteine) (6), O22-(7), or N (histidine) (1). The 570-nm band has been assigned as peroxide to copper charge transfer (1) or d-d transitions (6).We have undertaken a systematic study of a series of hemocyanin derivatives in order of increasing complexity to provide insight into the geometric and electronic structure of the active site of oxyhemocyanin (Fig. 1). The resulting spectral perturbations allow a determination of the oxidation state of the copper ions in each derivative, the existence of both exogenous and endogenous bridges between the two coppers, the coordination mode of peroxide in oxy, and a spectral assignment for the unique absorption features exhibited by oxyhemocyanin. MATERIALS AND METHODSBusycon canaliculatum hemocyanin was purified by ultracentrifugation. A Varian E-9 EPR spectrometer (X-band) was used on frozen solution samples. Low-temperature optical spectra were taken of buffered sucrose solutions with a Cary 17 absorption spectrometer and a Spectrim II cryocooler. Difference spectra of the 300-to 500-nm region were taken with the appropriate protein derivative in the reference beam-e.g., met apo-aquo provided the baseline for met apo-N3-. This was necessary to compensate for residual oxyhemocyanin and the steep slope of the protein absorption in the 300-to 500-nm region. RESULTS AND DISCUSSIONMet apo hemocyanin Met apo hemocyanin is prepared (8) The EPR spectrum of met apo-N3-is given in Fig. 2

Crystal structure of deoxygenated limulus polyphemus subunit II hemocyanin at 2.18 Å resolution: Clues for a mechanism for allosteric regulation

“…In addition, the cupric ions must be strongly antiferromagnetically coupled by the bound peroxide, since oxygenated hemocyanins are EPR silent (Dooley et al, 1978). Based on these data several models for the oxygenated dinuclear copper site in hemocyanin have been proposed (Eickman et al, 1979;Maddaluno & Giessner-Prettre, 1991;Ross & Solomon, 1991).…”

Electronic and geometric structure-function correlations of the coupled binuclear copper active site

“…The theme of this talk will be to first develop our chemical-spectroscopic approach [1,2,3] for one mollusc hemocyanin in order to elucidate the unique spectroscopic features associated with its coupled binuclear copper active site. These features will then be used to generate a "spectroscopically effective" picture [3] of this site.…”

Preparation of a spectral probe derivative of the hemocyanin biopolymer: effects of allosteric interactions on the coupled binuclear copper active site.

“…1 is reasonably well defined on the basis ofour chemical, spectral, and theoretical studies (11,12) of the series of derivatives shown in this figure, as well as resonance Raman studies, which indicated (19) the peroxide oxidation state of the bound dioxygen, and extended x-ray absorption fine structure (EX-AFS) analysis, which gave a Cu-Cu distance of 3.67 A (20) or 3.55 A (21). Cu(I) has a tendency toward lower coordination numbers, softer ligands, and a geometry based on ligand-ligand replusion (rather than unpaired d electron effects) relative to Cu(II).…”