We report reconstitution of photoactive yellow protein (PYP) from apoPYP and p-coumaric acid derivatives. The addition ofp-coumaric acid to the apoPYP sample did not result in the recovery of PYP. In contrast, yellow products were obtained by the addition of p-coumaryl thiophenyl ester or pcoumaric anhydride to the apoPYP sample, the absorption spectra of which were indistinguishable from the spectrum of intact PYP. Our findings provide strong evidence that PYP has the p-coumaryl chromophore. This reconstitution technique opens the way for further biophysical studies of PYP using artificial chromophore analogs.Key words'." Photoactive yellow protein; Photoreceptor protein; p-Coumaric acid; Chromophore; Reconstitution; Ectothiorhodospira halophila 1, IntroductionPhotoactive yellow protein (PYP) is present in Ectothiorhodospira halophila as a soluble small chromoprotein [1]. It has a broad absorption band, the absorption maximum of which is at 446 nm [1]. It is considered a photoreceptor protein for the negative phototaxis of E. halophila [2]. PYP has a photoreaction cycle comprised of several intermediates, the spectral and kinetic properties of which are very similar to those of the retinal proteins in halobacteria [3 5]. Formation of the bathochromic photoproduct occurs upon light absorption, the photoproduct being converted to a near-UV intermediate in a microsecond time scale and reverting to the ground state in a subsecond time scale. These intermediates correspond to the K (batho) or L (lumi) and M (meta) intermediates of the retinal proteins. The relation between PYP and the retinal proteins, therefore, is a matter of interest, but the nature of the PYP protein moiety is very different from the moieties of the retinal proteins [6,7]. Retinal proteins are membrane proteins composed of seven transmembrane helices [8,9] and have molecular weights of 24,000-40,000. PYP is a soluble small protein of 14 kDa which crystal structure analysis to 1.4-A resolution has shown to have the ot/fl-fold structure [7].The PYP chromophore first was thought to be retinal because its photochemical properties are similar to those of retinal proteins [10]. The chromophore-binding site was tentatively assigned to the lysine residue at the 111 position spectroscopy has shown that the molecular weight of the chromophore is 147, smaller than that of retinal [6]. Moreover, the chromophore binding site is a unique cysteine residue at position 69 [6]. These findings suggest that the chromophore is not retinal. Recent studies have shown that the PYP chromophore is p-coumaric acid (4-hydroxycinnamic acid) which binds to the cysteine residue via a thioester bond [11,12] (Fig. la,b).Although studies based on NMR analysis of the chromophore released from digested PYP [11] or high-resolution crystallography of PYP [12] clearly have shown that PYP has the p-coumaryl chromophore, direct confirmation would depend on the reconstitution of PYP from its apoprotein and pcoumaric acid. Moreover, an effective reconstitution technique would pr...

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We previously demonstrated that amilorides bind to the quinone binding pocket of bovine mitochondrial complex I, not to the hitherto suspected Na⁺/H⁺ antiporter-like subunits (ND2, ND4, and ND5) [Murai, M., et al. (2015) Biochemistry 54, 2739-2746]. To characterize the binding position of amilorides within the pocket in more detail, we conducted specific chemical labeling [alkynylation (-C≡CH)] of complex I via ligand-directed tosyl (LDT) chemistry using a newly synthesized amide-type amiloride AAT as a LDT chemistry reagent. The inhibitory potency of AAT, in terms of its IC50 value, was markedly higher (∼1000-fold) than that of prototypical guanidine-type amilorides such as commercially available EIPA and benzamil. Detailed proteomic analyses in combination with click chemistry revealed that the chemical labeling occurred at Asp160 of the 49 kDa subunit (49 kDa Asp160). This labeling was significantly suppressed in the presence of an excess amount of other amilorides or ordinary inhibitors such as quinazoline and acetogenin. Taking into consideration the fact that 49 kDa Asp160 was also specifically labeled by LDT chemistry reagents derived from acetogenin [Masuya, T., et al. (2014) Biochemistry 53, 2307-2317, 7816-7823], we found this aspartic acid to elicit very strong nucleophilicity in the local protein environment. The structural features of the quinone binding pocket in bovine complex I are discussed on the basis of this finding.

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