An anthocyanin pigment was first isolated in 1913 as a red oxonium salt from the blue cornflowerCentaurea 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, seesupplementary 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 (seesupplementary information).

a, Side view along the pseudo-three-fold axis. Blue, anthocyanin; yellow, flavone glycoside; spheres: red, Fe³⁺; green, Mg²⁺; black, Ca²⁺. For stereo view, seesupplementary 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.

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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.

Authors and Affiliations

1. Department of Physics, Graduate School of Science, Kyushu University, Hakozaki 6-10-1, Fukuoka, 812-8581, Japan

   Masaaki Shiono

2. Photon Factory, High Energy Accelerator Research Organization, Oho 1-1, Tsukuba, 305-0801, Japan

   Naohiro Matsugaki

3. Department of Biology, Tokyo Gakugei University, Koganei, Tokyo, 184-8501, Japan

   Kosaku Takeda

Corresponding author

Correspondence toKosaku Takeda.

Competing interests

The authors declare no competing financial interests.

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