ythe

N

y of

; acce 4

of andrea

successfully attached to cyt. c by a reaction between the quinone moiety of 1 and the amino group or guanidino group of cyt. c. Inthe reaction at pH 9, the ratio of cyt. c to 1 in the product was estimated to be 1:0.8 by UVvis spectroscopy. 2005 Elsevier B.V. All rights reserved.

Since pdon can interact via a diiminic binding site and

atom of pdon is liable to be attacked by a nucleophile.Indeed, many condensation reactions between an o-quinoid

plexes, have many attractive properties as chemical modi-

proteins. In this paper, the chemical modication of pro-teins by a ruthenium complex containing pdon was exam-ined in mild conditions.

The complex, [Ru(pdon)(bpy)2](ClO4)2 (bpy = 2,20-bipyr-

idyl) ([1](ClO4)2) (Scheme 1), was reacted with cytochromec (cyt. c) as a model protein.

* Corresponding author. Tel./fax: +81 42 388 7482.E-mail address: nobu1@cc.tuat.ac.jp (N. Nakamura).

Inorganic Chemistry Communicatthrough an o-quinoid group, it acts as a bridging ligandto construct binuclear or multinuclear complexes [59]. Inthese complexes, an o-quinoid moiety coordinates directlywith dierent metal centers. On the other hand, interactionbetween the carbon atom in the carbonyl moiety and theoxygen atom of the perchlorate anion was shown in thecrystal structures of the perchlorate complexes of ruthe-nium, manganese, and iron [10,11]. The crystal structureof an acetonyl-group-linked pdon complex was reportedby Tanaka et al. [12]. These results suggest that the carbon

ers of proteins. A series of ruthenium-modied proteins,each consisting of a well-designed ligand and mutant pro-tein, have been synthesized for the study of the electrontransfer process in proteins [19]. On the other hand, a facilemodication to proteins by the ruthenium complex isexpected to expand the application of this fascinating com-plex. Chemical modication using the ruthenium complexmight be applicable to other unknown proteins. Recently,Ueyama et al. [20] reported a bis(terpyridine) rutheniumderivative, which is a N-terminal modier of peptides andKeywords: Ru(II) complex; Phenanthroline quinone; Nucleophilic reaction; Cytochrome c

1,10-Phenanthroline-5,6-dione (pdon) is a chelate ligandcontaining an o-quinoid moiety which has many interestingcharacteristics. Owing to its redox activity, pdon in ametal-free state and in complexes with transition metals(ruthenium, cobalt, osmium, iron, and nickel) shows strongelectrocatalytic activity for the oxidation of NADH [14].

moiety of pdon and a diamine group have been reported[1318]. Lysine and arginine have a nucleophilic compo-nent in their side chains, thus, the amino group and guani-dino group of a protein might be allowed to react with thequinone moiety of pdon as a nucleophile.

Transition metal complexes, especially ruthenium com-Chemical modication of ccomplex containing p

Keiko Yokoyama, Takafumi Asakura,

Department of Biotechnology and Life Science, Tokyo Universit

Received 19 October 2005Available onlin

Abstract

The chemical modication of proteins by the quinone moietyusing [Ru(pdon)(bpy)2](ClO4)2 (bpy = 2,2

0-bipyridyl) ([1](ClO4)2) aa peak of cyt. c modied with 1. The reactivity of 1 for cyt. c inc1387-7003/$ - see front matter 2005 Elsevier B.V. All rights reserved.doi:10.1016/j.inoche.2005.11.019ochrome c by a rutheniumnanthroline quinone

obuhumi Nakamura *, Hiroyuki Ohno

Agriculture and Technology, Koganei, Tokyo 184-8588, Japan

epted 15 November 2005January 2006

1,10-phenanthroline-5,6-dione (pdon) complex was investigatedcytochrome c (cyt. c) at various pH (79). Mass spectra revealedsed with the pH of the solution. These results suggest that 1 was

www.elsevier.com/locate/inoche

ions 9 (2006) 281283

RuC32H22N6O2 = 624) was assembled on the protein.Since deconvolution took no account of the charge-statechange between an unmodied protein and a 1 cation(2+ charge state) modied protein, the value of 621 Dawas obtained by simply multiplying of each set of peaks.

a

b

Fig. 1. Mass spectra of native cyt. c (a) and the 1-modied cyt. c reacted atpH 9 (b) (Inset; Enlarged view of 8+ charged species.).

282 K. Yokoyama et al. / Inorganic Chemistry Communications 9 (2006) 281283The ligand pdon and [1](ClO4)2 were synthesized andpuried according to methods reported previously [10,21].Cyt. c (from horse heart) which has an acetylated N-termi-nus was obtained from Sigma Chemical Co. as a powder(M.W. 12384). For modication, cyt. c was mixed with 10equivalent of [1](ClO4)2 in 25 mM HEPES buer at variouspH (pH 79), and reacted at room temperature for threedays. The 1-modied proteins were washed with water sev-eral times in an amicon ultra-15 centrifugal lter device(Amicon) until the unreacted 1 in the ltrate was no longerdetectable using UVvis spectroscopy. The products wereanalyzed with an ESITOF MS spectrometer (JEOLJMS-T100LC). Deconvolution of the mass spectra was per-formed using ESI deconvolution Ver. 2.01 of JEOL. Theelectronic absorption spectra were measured at room tem-perature with a Shimadzu UV-2500PC spectrometer.

The mass spectrum of native cyt. c shows a series of ionswith a molecular mass of 12384 Da (Fig. 1a). The massspectrum of the product reacted in the buer solution atpH 9 revealed a series of additional ions (Fig. 1b). Thepeaks at m/z 1549.03, m/z 1626.88, and m/z 1704.35 areassigned to an 8+ charge state protein ([Cyt. c + 8H]8+ =12392; calc. m/z = 1549.00), the adduct of a 6+ chargestate protein and a cation 1 ([Cyt. c + (1) + 6H]8+ =13014: calc. m/z = 1626.75), and the adduct of a 4+ chargestate protein and two 1 cations ([Cyt. c + 2(1) + 4H]8+ =13636: calc. m/z = 1704.50), respectively. The guanidinoor amino group of cyt. c might attack the carbon atomof the quinone moiety of 1, subsequently forming theadduct of 1 and cyt. c (Scheme 2).

Fig. 2 shows deconvolution data of the mass spectra forthe products reacted at various pH (pH 79). All deconvo-lution spectra exhibited at least two peaks. One of them

Scheme 1.corresponded to unmodied cyt. c (12384 Da). The otherpeak was centered at 13005 Da which is 621 Da higherthan native cyt. c, indicating that a 1 cation (calc.

SchemeIt can thus be suggested that 1 successfully attached tocyt. c under these mild conditions. To be precise, the inten-sity of the peak of 13005 Da increases with the pH of the

2.

istryFig. 2. Deconvoluted data of all ions of 1-modied cyt. c reacted at pH 7(a), pH 8 (b), and pH 9 (c).

K. Yokoyama et al. / Inorganic Chembuer solution. Furthermore, a small peak at 13628 Dawas clearly visible in the deconvolution spectrum of theproduct reacted in the buer solution at pH 9, indicatingthat two cations of 1 were modied to cyt. c. Since aminomoieties and guanidino moieties in the side chain aredeprotonated at higher pH solution, the number of modi-ed cations was increased.

The UVvis spectra of native cyt. c, 1, and the productreacted at pH 9 are shown in Fig. 3. The spectrum of theproduct is a superposition of the spectrum of cyt. c andthat of 1. The ratio of cyt. c to 1 in the product was esti-mated to be 1:0.8. This value was calculated from theabsorbance of the band at 284 nm (e284 for cyt.c = 22500, e284 for 1 = 67800) and the band at 413 nm(e413 for cyt. c = 128000, e413 for 1 = 11600) [22]. Thisresult demonstrates that UVvis spectroscopy can be usedto estimate the number of 1 cations modied on a protein.

In conclusion, this paper described the reactivity of aquinone moiety of the ruthenium complex [1](ClO4)2 as afacile modier of proteins. The modication of the 1 cationto cytochrome c was conrmed by mass spectroscopy. The

Fig. 3. UVvis spectra of 1 (dashed line), cyt. c (dashed and dotted line),and 1-modied cyt. c (solid line).increase in reactivity at higher pH can account for thedeprotonation of amino groups or guanidino groups ofcyt. c. The ratio of cyt. c to 1 in the product reacted atpH 9 was estimated to be 1:0.8 based on UVvis spectros-copy. These results suggest that 1 might adapt to a simplemodication of a metal complex on a protein surface undermild conditions. Elucidation of the site where 1 attachedand optimal conditions for the modication is under way.

Acknowledgments

This study was supported by grants from the Ministry ofEducation, Science, Sports, Culture, and Technologythrough the Tokyo University of Agriculture and Technol-ogy as a part of the 21st century COE (Center of Excel-lence) program of the Future Nano-Materials researchand education project, the Japan Society for the Promotionof Science (No. 17550149 to N.N.).

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Chemical modification of cytochrome c by a ruthenium complex containing phenanthroline quinoneAcknowledgmentsReferences