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The reaction of nitrite at pH 5.0-7.0 with the deoxyhaemocyanin of a mollusc, the Roman snail (Helix pomatia), yielded nitrosylhaemocyanin (CuIA.NO+ CuIIB), in contrast with the formation of methaemocyanin with the deoxyhaemocyanin of the crustacean Astacus leptodactylus (mud crayfish). With Helix haemocyanin 1 NO was thereby liberated per active site, as shown by m.s., as against 2 NO with Astacus haemocyanin. Helix nitrosylhaemocyanin was characterized in c.d. by the negative extremum at 336 nm (CuIA.NO+) and by the mononuclear e.p.r. signal at g = 2 (CuIIB). Binuclear e.p.r. signals have been observed after the addition of nitrite to methaemocyanins. With Astacus methaemocyanin, no further reaction occurred, whereas with Helix methaemocyanin the mononuclear e.p.r. signal, characteristic for nitrosylhaemocyanin gradually appeared. This formation of Helix nitrosylhaemocyanin implicates the binding, most likely on CuIIA, of a second nitrite besides a bridging nitrite, so that a dismutation into NO and NO2 can occur there. A further dismutation of NO2 yields nitrite and nitrate. The formation of the latter was demonstrated by Raman spectrometry. The reaction rate of Helix methaemocyanin with nitrite decreased with increasing pH according to the Henderson-Hasselbalch equation with a pKa value of 6.77, attributed to a mu-aquo bridging ligand, which can be exchanged for nitrite, in equilibrium with a mu-hydroxo ligand which cannot. These data also favour the formulation of the final reaction product as nitrosylhaemocyanin instead of semi-methaemocyanin, with or without bound nitrite.

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