G. Alzuet et al., THE BINDING OF AZIDE TO COPPER-CONTAINING AND COBALT-CONTAINING FORMSOF HEMOCYANIN FROM THE MEDITERRANEAN CRAB CARCINUS-AESTUARII, European journal of biochemistry, 247(2), 1997, pp. 688-694
To establish the competence of the active site of hemocyanin to acquir
e diverse coordination geometries, the binding of azide to three forms
of a crab hemocyanin, the dinuclear cupric or met-hemocyanin, the mon
onuclear cupric or met-apo-hemocyanin. and the mononuclear Co(II)-subs
tituted derivative has been studied by near-ultraviolet circular dichr
oism and EPR spectroscopies. The near-ultraviolet circular dichroism s
pectra of the various derivatives present qualitatively similar featur
es, namely a negative peak around 335 nm in the case of the two copper
-containing derivatives and a three-component pattern with the Co(II)
derivative. Upon decreasing the pH from 7.0 to 5.5 a decrease of optic
al activity is observed with all protein samples. The characteristic C
D features, attributable to N(imidazole)-to-metal and to OH--to-metal
charge transfer transitions, are strongly affected by azide binding. I
n particular, the intensity of the negative band exhibited by the two
copper-containing protein forms decreases with the onset of a new nega
tive feature with maximum around 400 nm diagnostic for azide-to-Cu(II)
charge-transfer transitions. The visible region is affected as well,
indicating that changes in the coordination sphere of copper take plac
e. The affinity for azide of the different protein forms is higher at
low pH. EPR measurements on the paramagnetic met-apo-hemocyanin deriva
tive as a function of pH demonstrate heterogeneity in the coordination
environment at low pH. Tn the presence of azide an increase of rhombi
c distortion of the EPR spectra is observed and on the basis of the id
entified sets of copper hyperfine features in the course of azide titr
ation experiments two different azide bound forms of met-apo-hemocyani
n can be detected. The CD and EPR data at the different pH values are
consistent with a reaction scheme in which azide replaces a fourth lig
and in the metal-coordination sphere, identified as a water or hydroxi
de molecule.