Extraction of the functional manganese and calcium from photosystem II.

Manganese (Mn) and calcium (Ca) are both metal cofactors required for photosynthetic oxygen evolution. The functional roles for these ions in the O2-evolving reactions are not completely known. They are studied by comparative spectroscopic measurements between intact and metal-depleted samples. In this chapter, we outline three experimental procedures used for the various removal of Mn and Ca from photosystem (PS) II-containing (i.e,. O2-evolving) preparations: the complete Mn extraction using a strong alkaline Ches buffer (pH 9.4)/MgCl2 wash, partial Mn extraction using a mild hydroxylamine (pH 6.8) wash, and specific Ca extraction through a low pH/citrate (pH 3) wash. The O2 evolution activities (measured by a Clarke-type oxygen electrode), protein composition (determined by sodium dodecyl sulfate- polyacrylamide gel electrophoresis), and the relative Mn and Ca content (measured by atomic absorption spectroscopy) are reported for each extraction procedure.

(18)O isotope exchange measurements reveal that calcium is involved in the binding of one substrate-water molecule to the oxygen-evolving complex in photosystem II.

Direct evidence is presented to show that calcium is inherently involved in the binding of one of the two substrate-water molecules to the oxygen-evolving complex in photosystem II. Previous rapid (millisecond range) (18)O isotope exchange measurements between added H(2)(18)O and the photogenerated O(2) have shown that the two substrate-water molecules bind to separate sites throughout the S-state cycle, as revealed by their kinetically distinct rates of (18)O exchange [Hillier, W., and Wydrzynski, T. (2000) Biochemistry 39, 4399-4405]. Upon extraction of the functionally bound calcium using a either a low-pH/citrate treatment or a NaCl/A23187/EGTA treatment and subsequent reconstitution of activity with strontium, we show for the first time a specific increase in the slow rate of (18)O exchange by a factor of 3-4. This increase in the slow rate of exchange is consistently observed across the S(1), S(2), and S(3) states. In contrast, the fast phase of (18)O exchange in the S(3) state appears to be affected little upon strontium reconstitution, while the fast phases of exchange in the S(1) and S(2) states remain largely unresolvable, at the detectable limits of the current techniques. The results are discussed in terms of a possible substrate bridging structure between the functional calcium and a catalytic manganese ion that gives rise to the slowly exchanging component.

Amino acid deletions in the cytosolic domains of the chlorophyll a-binding protein CP47 slow Q(A)- oxidation and/or prevent the assembly of photosystem II.

The Photosystem II (PSII) core antenna chlorophyll a-binding protein, CP47, contains six membrane-spanning alpha-helices separated by five hydrophilic loops: A-E. To identify important hydrophilic cytosolic regions, oligonucleotide-directed mutagenesis was employed to introduce short segment deletions into loops B and D, and the C-terminal domain. Four strains carrying deletions of between three and five residues were created in loop B. Two strains, with deletions adjacent to helices II and III, did not assemble PSII; however, the mutants delta(F123-D125) and delta(R127-S131) remained photoautotrophic with near wild-type levels of assembled reaction centers. In contrast, all deletions introduced into loop D, connecting helices IV and V, failed to assemble significant levels of PSII and were obligate photoheterotrophic mutants. However, deletions in the C-terminal domain did not prevent the assembly of PSII reaction centers although the mutant delta(S471 -T473), with a deletion adjacent to helix V1, exhibited retarded Q(A)- oxidation kinetics and the PSII-specific herbicide, atrazine, bound less tightly in the delta(S471-T473) and delta(F475-D477) strains. Deletions in the C-terminal domain also created mutants with large protein aggregates that were recognized by an antibody raised against the PSII reaction center D1 protein. Low-temperature fluorescence emission spectra of photoautotrophic strains carrying deletions in either the C-terminal domain or loop B did not provide evidence for impaired energy transfer from the phycobilisomes to the PSII reaction center. The data therefore suggest an important structural role for loop D in the assembly of PSII and a potential interaction between the C-terminal domain of CP47 and the PSII reaction center that, when perturbed, results in photoinduced protein aggregates involving the D1 protein.

The two substrate-water molecules are already bound to the oxygen-evolving complex in the S2 state of photosystem II.

The first direct evidence which shows that both substrate-water molecules are bound to the O(2)-evolving catalytic site in the S(2) state of photosystem II (PSII) is presented. Rapid (18)O isotope exchange measurements between H(2)(18)O incubated in the S(2) state of PSII-enriched membrane samples and the photogenerated O(2) reveal a fast and a slow phase of exchange at m/e 34 (which measures the level of the (16)O(18)O product). The rate constant for the slow phase of exchange ((34)k(1)) equals 1.9 +/- 0.3 s(-1) at 10 degrees C, while the fast phase of exchange is unresolved by our current experimental setup ((34)k(2) >or= 175 s(-1)). The unresolvable fast phase has left open the possibility that the second substrate-water molecule binds to the catalytic site only after the formation of the S(3) state [Hillier, W., and Wydrzynski, T. (2000) Biochemistry 39, 4399-4405]. However, for PSII samples depleted of the 17 and 23 kDa extrinsic proteins (Ex-depleted PSII), two completely resolvable phases of (18)O exchange are observed in the S(2) state of the residual activity, with the following rate constants: (34)k(1) = 2.6 +/- 0.3 s(-1) and (34)k(2) = 120 +/- 14 s(-1) at 10 degrees C. Upon addition of 15 mM CaCl(2) to Ex-depleted PSII, the O(2) evolution activity increases to approximately 80% of the control level, while the two resolvable phases of exchange remain the same. In measurements of Ex-depleted PSII at m/e 36 (which measures the level of the (18)O(18)O product), only a single phase of exchange is observed in the S(2) state, with a rate constant ((36)k(1) = 2.5 +/- 0.2 s(-1)) that is identical to the slow rate of exchange in the m/e 34 data. Taken together, these results show that the fast phase of (18)O exchange is specifically slowed by the removal of the 17 and 23 kDa extrinsic proteins and that the two substrate-water molecules must be bound to independent sites already in the S(2) state. In contrast, the (18)O exchange behavior in the S(1) state of Ex-depleted PSII is no different from what is observed for the control, with or without the addition of CaCl(2). Since the fast phase of exchange in the S(1) state is unresolved (i.e., (34)k(2) > 100 s(-1)), the possibility remains that the second substrate-water molecule binds to the catalytic site only after the formation of the S(2) state. The role of the 17 and 23 kDa extrinsic proteins in establishing an asymmetric dielectric environment around the substrate binding sites is discussed.