J. Am. Chem. SOC. 1993,115, 11799-11805 11799 The Component of “Ruthenium Red” Responsible for Inhibition of Mitochondrial Calcium Ion Transport. Spectra, Electrochemistry, and Aquation Kinetics. Crystal Structure of Po- [( H C 0 2 ) ( N H 3 hRU12C13 Jeffrey Emerson,? M. J. Clarke,’J Wen-Long Ying? and D. Rao SanadP Contribution from the Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 021 67, and Department of Cell Physiology, Boston Biomedical Research Institute, 20 Staniford Street, Boston, Massachusetts 02114 Received August 17, 1993” Abstract: The cytological stain, ruthenium red, effectively inhibits mitochondrial Ca2+aq uptake; however, the majority of this activity has been attributed to an impurity. This component has now been isolated and a derivative, p-0- [(HCO~)(NH~)~RU]~C~~, characterized by X-ray diffraction, spectroscopy, and electrochemistry. The compound crystallizes in the orthorhombic space group Pnn2 (No. 34), with unit cell constants of a = 8.588 (l), b = 13.335 (2), c = 7.602 (2) A, 2 = 2, and R = 0.024. The bridging Ru-O bond is quite short (1.8240(6) A), while the Ru-Oformate bond length (2.033(3) A) is typical for a Ru-0 single bond. The equatorial ammineruthenium bond lengths average 2.1 l(2) A. The placement of the p-0 on a crystallographic two-fold axis dictates that the two metal centers are identical. The crystallographic equivalence together with electrochemicalevidence (Eo = 1.17 and 0.05 V) indicates strong electronic coupling between the mixed-valent ruthenium centers. The apical formates are easily replaced by chlorides in HCl. Aquation rates of both the formato and chloro complexes are pH dependent. For the formato complex kob is given by kobs = (kl[H+]’ + k2[H+]Ka1 + k3Ka1Ka2)/([H+I2 + [H+]Ka + KalKaz), where k~ = (7.8 f 0.1) X lW s-1, k2 = (1.2 f 0.1) X 10-4 s-l, k3 = -4 X 1W s-I, pKal = 2.45 f 0.07, and pKa2 = -9. The active species in solution appears to be the oxo-bridged ion, [X(NH3)4Ru-O-Ru(NH3)dXl3+, where X = C1- or OH-. The hydroxo- capped complex decomposes above pH 7. Theoretical calculationsshow these ions to be highly deformable, which may partially explain their ability to inhibit Ca2+binding to a number of different sites. The mixed-valent complex, ruthenium red, has reached its centennial of use as a cytological stain’ and has recently been widely investigatedas a drug interferingwith calcium u t i l i z a t i ~ n . ~ ~ ~ Originally employed to visualize pectins in plant cell walls by light microscopy, it is a generic stain for polyanions with high charge density, such as acidic mucopolysaccharides and DNA,4 and has been used for the past three decades as a selective stain for mitochondria and muscle fibrils in both visible and electron micro~copy.~ Its chemical and physiological properties have been previously summarized.6 Aside from its extensive histochemical use, ruthenium red is useful as a gel stain for a variety of Ca2+-binding proteins with different types of Ca2+-binding sites.’ It inhibits Ca2+binding to calmodulin3 and specifically inhibits uptake of Ca2+ by affecting both the Ca2+-pump and -releasemechanism.* Nanomolar levels of ruthenium red stronglyinhibit respiration-driven Ca2+, uptake in mitoch~ndria.~J~ Ruthenium red also blocks Ca2+ transport in other systems including the Ca2+ release channels of the sarcoplasmicreticulum,IlJ2 voltage-dependent Ca2+ channels in cisplatin-sensitive and -resistant leukemia cells,13 as well as Ca2+ f Boston College, Clarke@Hermes.BC.edu. t Boston Biomedical. 8 Abstract published in Advance ACS Abstracts, November 15, 1993. 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(22) Carrondo, M. A. A. F. d. C. T.; Griffith, W. P.; Hall, J. P.; Skapki, A. C. Biochem. Biophys. Acta 1980, 627, 332-334. (23) Luft, J. H. Anat. Rec. 1971, 171, 347-368. 1989,38, 1109-1 113. OOO2-7863/93/1515-11799$04.00/0 0 1993 American Chemical Society