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J. Biol. Chem., Vol. 280, Issue 13, 12371-12381, April 1, 2005

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Recruitment of a Foreign Quinone into the A₁ Site of Photosystem I

CHARACTERIZATION OF A menB rubA DOUBLE DELETION MUTANT IN SYNECHOCOCCUS SP. PCC 7002 DEVOID OF F_X, F_A, AND F_B AND CONTAINING PLASTOQUINONE OR EXCHANGED 9,10-ANTHRAQUINONE^*

Yumiko Sakuragi, Boris Zybailov, Gaozhong Shen, Donald A. Bryant, John H. Golbeck¶, Bruce A. Diner||, Irina Karygina**, Yulia Pushkar**, and Dietmar Stehlik**

From the Department of Biochemistry and Molecular Biology and Department of Chemistry, Pennsylvania State University, University Park, Pennsylvania 16802, ^||Central Research and Development, Experimental Station, E. I. du Pont de Nemours & Co., Wilmington, Delaware 19980-0173, and ^**Institut für Experimentalphysik, Freie Universität Berlin, Arnimallee 14, D-14195 Berlin, Germany

A photosystem I (PS I) complex containing plastoquinone-9 (PQ-9) but devoid of F_X, F_B, and F_A was isolated and characterized from a mutant strain of Synechococcus sp. PCC 7002 in which the menB and rubA genes were insertionally inactivated. In isolated PS I trimers, the decay of P700⁺ measured in the near-IR and the decay of A₁^– measured in the near-UV were found to be biphasic, with (averaged) room temperature lifetimes of 12 and 350 µs. The decay-associated spectra of both kinetic phases are characteristic of the oxidized minus reduced difference spectrum of a semiquinone, consistent with charge recombination between P700⁺ and PQ-^9–. The amplitude of the flash-induced absorbance changes in both the near-IR and the near-UV show that approximately one-half of the A₁ binding sites are either empty or nonfunctional. A spin-polarized chlorophyll triplet is observed by time-resolved EPR, and it is attributed to the ³P700 product of charge recombination via the T₀ spin level in those PS I complexes that do not contain a functional quinone. In those A₁ sites that are occupied, the P700⁺Q^– polarization pattern indicates that PQ-9 is oriented in a similar manner to that in the menB mutant. When excess 9,10-anthraquinone is added in vitro, it displaces PQ-9 and occupies the A₁ binding site more readily than in the menB mutant. This can be explained by a greater accessibility to the A₁ site in the menB rubA mutant due to the absence of F_X and the stromal ridge polypeptides. The relatively low binding affinity of 9,10-anthraquinone allows it to be readily removed from the A₁ site by washing. However, all A₁ sites are shown to bind napthoquinones with high affinity and thus are proven to be functionally competent in quinone binding. The ability to readily displace PQ-9 from the A₁ site makes the menB rubA mutant ideal for introducing novel quinones, particularly anthraquinones, into PS I.

Received for publication, November 16, 2004 , and in revised form, January 6, 2005.

^* This work is supported by National Science Foundation Grant MCB-0117079 (to J. H. G.) and MCB 0077586 (to D. A. B.), United States Department of Agriculture Grant NRICGP 97-35306-4882 (to B. A. D.), and Deutsche Forschungsgemeinschaft Grant Sfb 498 A3 (to D. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. Tel.: 814-865-1163; Fax: 814-863-7024; E-mail: jhg5{at}psu.edu.

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