Uclacyanins, stellacyanins, and plantacyanins are distinct subfamilies of phytocyanins: Plant‐specific mononuclear blue copper proteins

Abstract

The cDNAs encoding plantacyanin from spinach were isolated and characterized. In addition, four new cDNA sequences from Arubidopsis ESTs were identified that encode polypeptides resembling phytocyanins, plant‐specific proteins constituting a distinct family of mononuclear blue copper proteins. One of them encodes plantacyanin from Arubidopsis. while three others, designated as uclacyanin 1, 2, and 3, encode protein precursors that are closely related to precursors of stellacyanins and a blue copper protein from pea pods. Comparative analyses with known phytocyanins allow further classification of these proteins into three distinct subfamilies designated as uclacyanins, stellacyanins, and plantacyanins. This specification is based on (1) their spectroscopic properties, (2) their glycosylation state, (3) the domain organization of their precursors, and (4) their copper‐binding amino acids. The recombinant copper binding domain of Arubidopsis uclacyanin I was expressed, purified, and shown to bind a copper atom in a fashion known as “blue” or type 1. The mutant of cucumber stellacyanin in which the glutamine axial ligand was substituted by a methionine (Q99M) was purified and shown to possess spectroscopic properties similar to uclacyanin I rather than to plantacyanins. Its redox potential was determined by cyclic voltammetry to be +420 mV, a value that is significantly higher than that determined for the wild‐type protein (+260 mV). The available structural data suggest that stellacyanins (and possibly other phytocyanins) might not be diffusible electron‐transfer proteins participating in long‐range electron‐transfer processes. Conceivably, they are involved in redox reactions occurring during primary defense responses in plants and/or in lignin formation.