Structure of the blue cornflower pigment

Packaging red-rose anthocyanin as part of a ‘superpigment’ in another flower turns it brilliant blue.

Abstract

The same anthocyanin pigment makes roses red but cornflowers blue¹, a phenomenon that has so far not been entirely explained. Here we describe the X-ray crystal structure of the cornflower pigment, which reveals that its blue colour arises from a complex of six molecules each of anthocyanin and flavone, with one ferric iron, one magnesium and two calcium ions. We believe that this tetrametal complex may represent a previously undiscovered type of supermolecular pigment.

Main

An anthocyanin pigment was first isolated in 1913 as a red oxonium salt from the blue cornflower Centaurea cyanus², and later the same pigment was discovered in a red rose³. The colour variation was variously ascribed to a difference in flower-petal pH (ref. 3), an association of the pigment with metal ions⁴ or with another pigment⁵. However, the blue-cornflower pigment (later named protocyanin^6,7) was found to contain iron and magnesium^8,9 in complex with anthocyanin and flavone^10,11; calcium is also essential for the reconstruction of protocyanin¹².

We determined the crystal structure of reconstructed protocyanin at 1.05 Å resolution. (For methods, see supplementary information.) In the refined molecule, the four metal ions Fe³⁺, Mg²⁺ and two Ca²⁺ align along the pseudo-three-fold axis (Fig. 1a) and are coordinated to six anthocyanin (cyanidin 3-O-(6-O-succinyl glucoside)-5-O-glucoside) and six flavone (apigenin 7-O-glucuronide-4′-O-(6-O-malonyl-glucoside)) molecules. The inner Fe³⁺ and Mg²⁺ ions are each coordinated to three anthocyanin molecules, respectively, and the two outer Ca²⁺ ions are each coordinated to three flavone molecules (see supplementary information).

Figure 1: Crystal structure of the protocyanin molecule.

a, Side view along the pseudo-three-fold axis. Blue, anthocyanin; yellow, flavone glycoside; spheres: red, Fe³⁺; green, Mg²⁺; black, Ca²⁺. For stereo view, see supplementary information. b, Side views of stacking pigment pairs. Left, one anthocyanin molecule binds Fe³⁺ (red), the other binds Mg²⁺ (green); centre, two flavones each bind to one Ca²⁺ (black); right, flavone and anthocyanin molecules bind to Ca²⁺ and Mg²⁺, respectively.

The two sites of the central nuclei have the same electron density, which corresponds to the average for Fe³⁺ and Mg²⁺ (results not shown). This suggests that the sites are occupied by either Fe³⁺ or Mg²⁺ because of the random orientation of the molecule along the pseudo-three-fold axis. To verify this, we investigated the X-ray structures of metal-substituted FeMgBa-, FeMnBa- and FeCdBa-protocyanins (results not shown). (Barium derivatives are more stable than calcium derivatives for isolation as crystals.)

In the FeMgBa-protocyanin, the electron density of the inner nuclei is almost the same as in protocyanin, whereas in the FeMnBa- and FeCdBa-protocyanins it is the average of Fe³⁺ and of the substituted ions, respectively. The distances between the two metals and the coordinating oxygen atoms vary according to the radii of the substituted metals. These results indicate that the two metals are heterogeneous. We conclude that the inner nuclei of protocyanin consist of Fe³⁺ and Mg²⁺ ions.

In the protocyanin molecule, the anthocyanins and flavones self-associate in pairs. The Fe³⁺ and Mg²⁺ ions are coordinated to different anthocyanin fragments in an anthocyanin pair (Fig. 1b, left); the two Ca²⁺ ions are coordinated with separate flavone fragments in a flavone pair (Fig. 1b, centre). Stacking of anthocyanin and flavone (co-pigmentation) is also evident (Fig. 1b, right). The C–C and C–O bond lengths in the B-ring of anthocyanin (data not shown) indicate that the anthocyanin is in the 4′-keto-quinoidal form.

Blue flower colours are caused mainly by delphinidin-type anthocyanins — for example, commelinin¹³. In protocyanin, however, the blue colour is produced by a cyanidin-type anthocyanin. The chelation of Fe³⁺ and Mg²⁺ with the 4′-keto-quinoidal base of anthocyanin is important for the blueing in protocyanin. The two Ca²⁺ ions coordinate with the flavones to form components that bring about co-pigmentation as well as stabilization of the molecule. The blue colour in protocyanin is therefore developed by a tetranuclear metal complex, which may represent a new type of supermolecular pigment.