Cytochrome (cyt) b559 has been proposed to play an important role in the cyclic electron flow processes that protect photosystem II (PSII) from light-induced damage during photoinhibitory conditions. However, the exact role(s) of cyt b559 in the cyclic electron transfer pathway(s) in PSII remains unclear. To study the exact role(s) of cyt b559, we have constructed a series of site-directed mutants, each carrying a single amino acid substitution of one of the heme axial-ligands, in the cyanobacterium Synechocystis sp. PCC6803. In these mutants, His-22 of the alpha or the beta subunit of cyt b559 was replaced with either Met, Glu, Tyr, Lys, Arg, Cys or Gln. On the basis of oxygen-evolution and chlorophyll a fluorescence measurements, we found that, among all mutants that were constructed, only the H22Kalpha mutant grew photoautotrophically, and accumulated stable PSII reaction centers ( approximately 81% compared to wild-type cells). In addition, we isolated one pseudorevertant of the H22Ybeta mutant that regained the ability to grow photoautotrophically and to assemble stable PSII reaction centers ( approximately 79% compared to wild-type cells). On the basis of 77 K fluorescence emission measurements, we found that energy transfer from the phycobilisomes to PSII reaction centers was uncoupled in those cyt b559 mutants that assembled little or no stable PSII. Furthermore, on the basis of immunoblot analyses, we found that in thylakoid membranes of cyt b559 mutants that assembled little or no PSII, the amounts of the D1, D2, cyt b559alpha and beta polypeptides were very low or undetectable but their CP47 and PsaC polypeptides were accumulated to the wild-type level. We also found that the amounts of cyt b559beta polypeptide were significantly increased (larger than two folds) in thylakoid membranes of cyt b559 H22YbetaPS+ mutant cells. We suspected that the increase in the amounts of cyt b559 H22YbetaPS+ mutant polypeptides in thylakoid membranes might facilitate the assembly of functional PSII in cyt b559 H22YbetaPS+ mutant cells. Moreover, we found that isolated His-tagged PSII particles from H22Kalpha mutant cells gave rise to redox-induced optical absorption difference spectra of cyt b559. Therefore, our results concluded that significant fractions of H22Kalpha mutant PSII particles retained the heme of cyt b559. Finally, this work is the first report of cyt b559 mutants having substitutions of an axial heme-ligands that retain the ability to grow photoautotrophically and to assemble stable PSII reaction centers. These two cyt b559 mutants (H22Kalpha and H22YbetaPS+) and their PSII reaction centers will be very suitable for further biophysical and biochemical studies of the functional role(s) of cyt b559 in PSII.

2 is one of the essential components of the photosystem II (PSII) in higher plants, green algae, and cyanobacteria (1-5). cyt b 559 is a heme-bridged heterodimer protein that is composed of one ␣-and one ␤-subunit (encoded by the psbE and psbF genes) of 9 and 4 kDa, respectively. Each subunit provides a His ligand (His-22 residue of ␣-or ␤-subunit of cyt b 559 ) for the non-covalently bound heme. In addition, cyt b 559 exhibits different redox potential forms; a high potential form with a midpoint redox potential around ϩ400 mV, an intermediate potential form around ϩ200 -250 mV, and a low potential form with a midpoint redox potential about ϩ50 -100 mV (Refs. 5-9 and references therein). Several previous studies have proposed that cyt b 559 participates in secondary electron transfer pathways, which protect PSII from photoinhibition (5, 10 -12). In these models the high potential form of cyt b 559 might donate its electron to reduce highly oxidized chlorophyll radical species generated in PSII reaction centers under the donor-side photoinhibitory conditions. On the other hand, cyt b 559 might accept an electron from the acceptor side of PSII (Q B , PQ, or pheophytin (primary pheophitin a electron acceptor)) from generating damaging singlet oxygen species under the acceptor-side photoinhibitory conditions (13-15). In addition, a novel quinonebinding site (Q C ) was identified in proximity to cyt b 559 in the new 2.9Å PSII crystal structure (4). The occupancy of this Q C site has been proposed to modulate the redox equilibration between cyt b 559 and the PQ pool (16,17) or to involve in the exchange of PQ on the Q B site from the pool (4). Despite the recent progress in understanding the structure and function of PSII, the exact function of cyt b 559 in PSII remains unclear.Prior mutagenesis studies with Synechocystis sp. PCC6803, Chlamydomonas reinhardtii, or Nicotiana tabacum showed no stable PSII reaction centers assembled in the absence of either cyt b 559 subunit (18 -24). In addition, an early site-directed mutagenesis study with Synechocystis 6803 showed that substituting either of the heme axial ligands (His-22 of the ␣-subunit or His-22 of the ␤-subunit) with Leu severely diminished the assembly or stability of PSII (23). Furthermore, another sitedirected mutagenesis study involving C. reinhardtii showed that the H22Y and H22M mutants of the cyt b 559 ␣ subunit accumulated 10 -15% of the PSII (compared with wild-type cells) and contained a disrupted heme pocket, whereas still retaining significant amounts of O 2 evolution activity (24). This

We identified a spontaneously generated mutant from Synechocystis sp. PCC6803 wild-type cells grown in BG-11 agar plates containing 5 mM Glu and 10 microM DCMU. This mutant carries an R7L mutation on the alpha-subunit of cyt b559 in photosystem II (PSII). In the recent 2.9 A PSII crystal structural model, the side chain of this arginine residue is in close contact with the heme propionates of cyt b559. We called this mutant WR7Lalpha cyt b559. This mutant grew at about the same rate as wild-type cells under photoautotrophical conditions but grew faster than wild-type cells under photoheterotrophical conditions. In addition, 77 K fluorescence and 295 K chlorophyll a fluorescence spectral results indicated that the energy delivery from phycobilisomes to PSII reaction centers was partially inhibited or uncoupled in this mutant. Moreover, WR7Lalpha cyt b559 mutant cells were more susceptible to photoinhibition than wild-type cells under high light conditions. Furthermore, our EPR results indicated that in a significant fraction of mutant reaction centers, the R7Lalpha cyt b559 mutation induced the displacement of one of the axial histidine ligands to the heme of cyt b559. On the basis of these results, we propose that the Arg7Leu mutation on the alpha-subunit of cyt b559 alters the interaction between the APC core complex and PSII reaction centers, which reduces energy delivery from the antenna to the reaction center and thus protects mutant cells from DCMU-induced photo-oxidative stress.