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Mutations D1-D59N and D1-D61E in the putative Ca2+-binding lumenal interhelical a-b loop of the photosystem II (PSII) D1 protein [Chu, H. A., Nguyen, A. P., and Debus (1995), Biochemistry 34, 5839-5858] were further characterized in terms of S-state cycling and photoactivation. Bare platinum electrode measurements of centrifugally deposited O2-evolving membranes isolated from the a-b loop mutants demonstrated a retarded appearance of O2 following single turnover flashes, although not to the extent of retardation seen in the Deltapsb0 mutant, which lacks the extrinsic manganese-stabilizing protein (MSP). Double flash measurements indicate that retarded O2 release in mutants coincides with a decrease in overall PSII turnover during the S3-[S4]-S0 transition. S2 and S3 decay measurements in the isolated membranes indicate that D1-D59N and D1-D61E have faster decays of these higher S-states in contrast to slowed decays in the Deltapsb0 mutant. Measurements of the flash interval dependence of photoactivation indicate that intermediates of photoactivation [light-dependent assembly of the (Mn)4 complex] are highly destabilized in the a-b loop mutants compared to both DeltapsbO and the wild-type: flash intervals of greater than 2 s result in the nearly complete decay of unstable photointermediate(s) in the D1-D59N and D1-D61E samples, whereas a similar loss does not occur until intervals even greater than 10 s in the DeltapsbO and wild-type samples. These results are consistent with a role for the residues D1-D59 and D1-D61 in modulating the redox properties of the higher S-states and, also, possibly in the binding the calcium ion involved in photoactivation.

The effects of La(3+) on the extracellular signal-regulated kinase (ERK) signaling were investigated to explore the mechanism by which La(3+) results in cell proliferation associated with apoptosis in mouse embryo fibroblast NIH 3T3 cells. Our data showed that La(3+) ions could induce a pulse of phosphorylation of ERK mainly through an unknown metal-sensing mechanism, which is different from the Ca(2+)-sensing receptor . The putative sensor protein showed one binding site for La(3+) with a dissociation constant of approximately 8 nM. Inductions of c-fos, c-myc, and cyclin D1 and phosphorylation of retinoblastoma protein (pRb) were observed after activation of ERK. These results are consistent with our previous observation that La(3+) promotes proliferation by helping the cells pass through the G1/S restriction point and enter S phase. This La(3+)-induced signaling cascade exhibited abnormally sustained c-myc induction and pRb phosphorylation. Furthermore, a continual increase of the p53 level was observed along with the signal transduction, and a significant decrease of B-cell lymphoma/leukemia-2 gene was observed after approximately 18 h of incubation. All of the results were highly correlated with the increase of S-phase population and apoptotic cells. Therefore, the experimental results suggested that La(3+) induced cell proliferation and apoptosis compatible to a p53-related mechanism in NIH 3T3 cells via an ERK-signaling cascade induced by a metal-sensing mechanism.

The functions of cytochrome c-550 and a 12 kDa protein in cyanobacterial oxygen evolution were studied with directed deletion mutants delta psbV and delta psbU of Synechocystis sp. PCC 6803, and the following results were obtained. (1) In contrast to the delta psbU mutant which is capable of autotrophic growth in the absence of Ca2+ or Cl- at a reduced rate, the delta psbV mutant lacking cytochrome c-550 could not grow at all without Ca2+ or Cl-. (2) The delta psbV mutant had a significantly reduced thermoluminescence emission intensity and flash oxygen yield, whereas the delta psbU mutant showed slight decreases in thermoluminescence intensity and flash oxygen yield, indicating corresponding decreases in the concentrations of O2-evolving centers in these mutants. (3) The delta psbV and delta psbU mutants exhibited elevated peak temperature for the thermoluminescence B- and Q-bands indicative of more stable S2 states. (4) The rise time of the O2 signal during the S3-[S4]-S0 transition was increased slightly in the delta psbV mutant but not in the delta psbU mutant. (5) The oxygen evolution was inactivated in the dark rapidly in the delta psbV mutant with a half-time of 28 min, but this did not happen in the delta psbU mutant. (6) Photoactivation of the oxygen-evolving complex after removal of the manganese cluster by hydroxylamine showed a higher quantum yield in the delta psbV mutant than in the delta psbU mutant or wild type. Taken together, these results indicated that cytochrome c-550 plays a substantial role in maintaining the stability and function of the manganese cluster in algal photosystem II, whereas the 12 kDa protein plays primarily a regulatory role in maintaining normal S-state transitions. These functional features of cytochrome c-550 and the 12 kDa protein were compared with those of the 23 and 17 kDa proteins in higher plant photosystem II and of the 33 kDa protein in both algal and plant photosystem II.

Photoactivation is the light-dependent ligation of Mn2+ into the H2O oxidation complex of photosystem II (PSII) and culminates in the formation of an enzymatically active complex containing Ca2+ and four Mn>/=3+. Previous kinetic analysis demonstrated that the genetic removal of the extrinsic manganese-stabilizing protein (MSP) increases the quantum yield of photoactivation 4-fold relative to that of the wild type, consistent with the hypothesis that MSP hinders access of Mn2+ to a site of photoligation [Burnap, R. L., et al. (1996) Biochemistry35, 874-882]. In this report, several Synechocystis sp. PCC6803 mutants with defined amino acid substitutions in the N-terminal region of MSP or the e-loop of intrinsic PSII protein CP47 [Putnam-Evans, C., et al. (1996) Biochemistry 35, 4046-4053] were characterized in terms of the binding of MSP to the intrinsic portion of the PSII complex and in terms of photoactivation kinetics. The charge-pair switch mutation, Arg384Arg385 --> Glu384Glu385 in the lumenal e-loop of CP47 (CP47 RR384385EE), exhibited the most severe impairment of MSP binding, whereas the Arg384Arg385 --> Gly384Gly385 (CP47 RR384385GG) mutation caused a more moderate impairment in binding. Single-substitution mutations at the highly conserved Asp9 or Asp10 positions in the amino-terminal region of MSP also resulted in a reduced binding affinity, but not as severe as that in CP47 RR384385EE. The relative quantum yield of photoactivation of hydroxylamine-extracted mutant PSII was generally found to correlate with the degree of MSP binding impairment, with the CP47 RR384385 mutants exhibiting the highest quantum yields. A two-locus, double-mutant construct involving deletion of MSP in the CP47 RR384385EE background was found to be only slightly more impaired in H2O oxidation activity than either of the corresponding single-locus mutant derivatives, indicating that mutations at these genetically separate loci encode physically interacting products affecting the same reaction parameter during H2O oxidation. Taken together, the results reinforce the concept that MSP interacts with the e-loop of CP47 at Arg384Arg385 and that disruption of this interaction causes significant alterations of the site of H2O oxidation in terms of assembly and enzymatic activity of the Mn cluster.

Dark deactivation and photoactivation of H2O-splitting activity were examined in a directed mutant (delta psbO) of Synechocystis sp. PCC6803 lacking the extrinsic manganese-stabilizing protein of the photosystem II (PSII) reaction center complex. Rapid (t1/2 = 10 min) losses of H2O-splitting activity were observed for delta psbO cells kept in the dark, but not for wild-type cells. The loss of H2O-splitting activity by delta psbO cells was suppressed by maintaining the cells under illumination and dark losses were rapidly (t1/2 < 1 min) reversed by light. Photoactivation kinetics of delta psbO and wild-type cells were compared following hydroxylamine extraction of PSII Mn. Photoactivation of delta psbO cells under continuous illumination occurs at an intrinsically faster rate (about 4-fold) than the wild-type. Virtually all of the increase in the rate of photoactivation can be accounted for by a corresponding 4-fold increase in the relative quantum yield of photoactivation as indicated by the yield of photoactivation as a function of flash number. The flash frequency dependence of photoactivation indicates a multi-quantum process in the mutant resembling the wild-type, but with significant increases in yields at all flash frequencies examined. The higher quantum yield of photoactivation in delta psbO cells occurs in the absence of large changes in the kinetics of the rate-limiting dark rearrangement. The results are consistent with increased accessibility (or affinity) and photooxidation of Mn2+ at one or both of the two binding sites involved in the initial stages of the photoactivation mechanism. In the context of previous results, it is proposed that MSP regulates the binding/photooxidation of the second Mn2+ of the photoligation sequence, but not the first.

Site-directed mutations were produced at three highly conserved amino acid positions of MSP of the photosystem II (PSII) reaction center in the transformable cyanobacterium Synechocystis sp. PCC6803. The highly conserved aspartate at position 9 of the mature MSP was changed to a lysine (strain MSP-D9K) to assess its role in the proposed N-terminal binding region (Eaton-Rye & Murata, 1989; Odom & Bricker, 1992). This strain accumulates normal levels of MSP, and the properties of the H2O-splitting enzyme are only slightly altered relative to the wild-type. In contrast, replacement of cysteine 20 with a serine, which is proposed to disrupt the intramolecular disulfide bridge (Tanaka et al., 1989), produces a phenotype with no detectable accumulation of MSP, despite normal levels of mRNA transcripts. Like the psbO deletion strain, the MSP-C20S mutant exhibits impaired O2 evolution activity and a greater stability of the S2 state as measured by thermoluminescence. Mutation of strictly conserved aspartate 159 to asparagine (MSP-D159N) does not affect the accumulation of MSP, but causes a reduction in the H2O-oxidation activity and thermoluminescence properties intermediate between the wild-type and the psbO deletion strain. In addition, we report upon improved methods for obtaining oxygen-evolving membranes from mutant cells.

CC-1065 is a potent antitumor antibiotic which bonds to duplex DNA specifically; the biological effects of the drug are presumably the consequences of its DNA interactions. In order to investigate the factors which may affect drug-DNA bonding in cells, a method using a thermal-alkaline treatment to induce phosphodiester bond breakage at the drug-DNA bonding sites and Southern DNA transfer-hybridization to quantify drug-DNA bonding at defined sequences in drug-treated cultured mammalian cells was developed. We have found that in vivo, in cultured Chinese hamster ovary (CHO) cells, CC-1065 bonds twice as efficiently in the highly amplified dihydrofolate reductase (DHFR) gene domains as in the nonamplified adenine phosphoribosyltransferase (APRT) gene domain. However, in vitro, in purified CHO cellular DNA, CC-1065 bonds equally to both the DHFR and APRT genes. We observed a significant degree of "gene-specific" preferential repair for drug-DNA adducts in the amplified DHFR gene domains, and it appears that this "gene-specific" repair reflects "transcribed-strand specific" repair. These results suggest that DNA amplification may affect drug-DNA adduct formation and transcription may affect its repair.