The reaction of nitrogen monoxide with the haemocyanins of the crayfish Astacus leptodactylus and the snail Helix pomatia

Abstract

The rate of the reaction of Astacus leptodactylus methaemocyanin with NO follows the Henderson-Hasselbalch equation with a pKa of 5.85, suggesting that one imidazole ligand of Cu was exchanged for NO. The reaction is blocked by F- as a bridging ligand. The same imidazole residue might be responsible for the decomposition of nitrosylhaemocyanin, [Cu1NO+CuII], with an unlocated binding site for NO, into methaemocyanin and NO, as the rate increase with pH. NO could react preferentially with CuA of Helix pomatia methaemocyanin, CuA'IICuBII, as it possibly has only two histidine ligands instead of the three of CuA in Astacus haemocyanin. This difference might explain the higher formation rate and the much greater stability of Helix nitrosylhaemocyanin. The fast reaction is governed by a pKa of 6.80, probably of a bridging mu-aquo ligand. With F- or a mu-hydroxo bridging ligand a low reaction rate was still observed, in contrast with Astacus methaemocyanin. Helix nitrosylhaemocyanin was transformed by N3- into methaemocyanin with the liberation of N2 and N2O. This methaemocyanin could almost quantitatively be regenerated with H2O2. Helix nitrosylhaemocyanin was only partially regenerated by a direct treatment with H2O2 and almost quantitatively by HONH2 in a similar fairly fast reaction, followed by a much slower one.

The reaction of nitrite with the haemocyanin of the Roman snail (Helix pomatia)

“…data, in the copper sites of Helix nitrosylHc, was already suggested by the liberation of only 1 NO per active site in the reaction of NO2with deoxyHc against 2 NO with Astacus deoxyHc, which yielded metHc (Tahon et al, 1988). This presence of NO was confirmed by the production of N2 and N20 in the reaction of N3-with Helix nitrosylHc (Tahon et al, 1989b) and here by the nitrite determinations with the Griess-Ilosvay reagent after acid decomposition of nitrosylHc.…”

“…and e.p.r. measurements_ shQwed their quantitative transformation into nitr6sy1Hc, with the exception of functional units g and h, which were only transformed to the extent of 68 and 41 % respectively (Tahon et al, 1989a). After 2 h reaction in a closed vessel of NaNO2 with Helix deoxyHc at pH 5.7, the gaseous reaction products were determined by m.s.…”

“…3) would require a very high Eo for the remaining Cu(I). The formation of nitrosylHc on treating Astacus and Helix metHc with NO (Tahon et al, 1989b) and its slow formation in the reaction of Helix metHc with nitrite cannot be explained by the reduction of one Cu(II) by NO and by NO2-(eqn. 6), for this would imply an oxidation of NO to NO2and of NO2to NO2.…”

“…The pH-dependence of the formation of nitrosylHc in the reaction of Astacus deoxyHc with NO was interpreted by the detachment and protonation of one imidazole ligand. The deprotonation of this imidazole residue might inversely be responsible for the lower stability of Astacus nitrosylHc (Tahon et al, 1989b).…”