George M. Cheniae

Photoactivation of the water-oxidizing complex in Photosystem II membranes depleted of Mn and extrinsic proteins. I. Biochemical and kinetic characterization

Abstract Conditions were defined for obtaining photoligation of Mn 2+ and photoactivation of O 2 evolution in NH 2 OH- and Tris-extracted PS II membranes (TMF-2) completely devoid of the CF 0 /CF 1 complex and containing only approx. 2–3 PS II e − acceptor equivalents per PS II reaction center. At optiomal pH (pH 6.5) only light and Mn 2+ were essential for Mn ligation by the apo-S-state complex (approx. 4 Mn/reaction center); however, Ca 2+ addition was required for maximal expression of water oxidation activity of the photoligated Mn. In the absence of added PS II e − acceptors, the quantum efficiency and yield of photoactivation was diminished and was insensitive to atrazine. PS II e − acceptors increased the quantum efficiency/yield by approx. 2-fold and conferred sensitivity to atrazine. Kinetic analyses of the photoactivation process gave evidence for a rate constraint ( t 1 2 ≈ 125 ms ) and an unstable intermediate (half-life of approx. 1.0 s). Cl − was not absolutely essential for photoactivation. A 7-fold increase of rate of O 2 evolution was obtained without Cl − addition to Cl − -depleted NH 2 OH-TMF-2; Cl − addition during photoactivation gave only a 2-fold additional increase. This Cl − effect ( K m = 3.8 mM) was different from the Cl effect ( K m = 1.5 mM) on O 2 evolution. Weak light ageing of NH 2 OH-TMF-2 in the absence (but not in the presence) of Mn 2+ inhibited photoactivation and photoreduction of DCIP by Mn 2+ ; however, DCIP photoreduction by DPC or TPB was not diminished. Such weak light ageing also increased the VLP-form of Cyt b -559, but there was no apparent correlation between the increase in O 2 evolution and the conversion of the VLP- and LP-Cyt b -559 to the HP-Cyt b -559 during photoactivation. Photoactivation is suggested to be a two-quantum process in which two Mn 2+ are sequentially bound, photooxidized and ligated by the apo-S-state complex. This process facilitates ligation of two additional Mn ions to form the tetra-Mn S-state water-oxidizing complex.

Photoactivation of the water-oxidizing complex in photosystem II membranes depleted of Mn, Ca and extrinsic proteins. II: Studies on the functions of Ca2+

The effects of Ca 2+ addition to PS II membranes depleted of Ca, Mn and the 17/23 kDa proteins (NH 2 OH-TMF-2) on photoactivation of the water-oxidizing Mn-complex were studied. Lightincubation of NH 2 OH-TMF-2 with MnCl 2 but no Ca 2+ resulted in an increase of EDTA non-extractable membrane-bound Mn abundance; however, the resulting Mn-complex was inactive in O 2 evolution. Post-addition and incubation of Ca 2+ with these membranes in darkness conferred catalytic water-oxidizing activity to the membranes and caused release of one or two membrane-bound Mn atoms per reaction center. Among divalent cations, the hierarchy of effectiveness for conferring activity was Ca 2+ > Sr 2+ ≫ Ba 2+ ,Mg 2+ , a result similar to the cation specificity for reconstitution of water-oxidizing activity of PS II membranes depleted of Ca 2+ and 17/23 kDa proteins (Ghanotakis, D.F., Babcock, G.T. and Yocum, C.F. (1984) FEBS Lett. 167, 127–130). Whereas NH 2 OH-TMF-2 photoactivated in Mn 2+ /Ca 2+ exhibited a typical period-four oscillation of thermoluminescence B-band, membranes photoactivated in Mn 2+ only showed a high yield of B-band with an upshifted peak temperature aftera single flash but no subsequent oscillatory behavior. Post-incubation in darkness with divalent cations (effectiviry of Ca 2+ > Sr 2+ ≫ Ba 2+ ,Mg 2+ ) permitted the subsequent period-four oscillation of the B-band. These results indicate: (1) photoligation of Mn 2+ into the Mn-complex does not require Ca 2+ addition to NH 2 OH-TMF-2; (2) the Mn-complex formed during photoactivation in the absence of Ca 2+ are inactive in water oxidation, a consequence of an inhibition of the S 2 → S 3 transition; and (3) inactive complexes are converted to catalytically active complexes by dark incubation with Ca 2+ . In analyses for the thermoluminescence D-band (S 2 Q − A with NH 2 OH-TMF-2 photoactivated in Mn 2+ only, the normal D-band and an additional emission band with a high peak temperature of 45 °C were observed. Dark incubation with Ca 2+ before illumination to populate S 2 Q − A charge pair diminished the 45 °C emission without a proportionate increase of the normal D-band.

Effects of Photosystem II extrinsic proteins on microstructure of the oxygen-evolving complex and its reactivity to water analogs

Abstract We used Triton-prepared PS II membranes in studies of the inactivation of O2 evolution and solubilization of Mn and specific PS II polypeptides by NH2OH, N- and O-substituted NH2OH derivatives, NH2NH2 and NH4Cl. The inactivation of O2-evolution, solubilization of Mn and the solubilization of the extrinsic PS II polypeptides (17, 23 and 33 kDa) proved closely correlated, half-maximal effects occurring with only 100 μM NH2OH. NH2OH (2 mM) and NaCl (1 M) extractions solubilized about one-half the amount of protein solubilized by 0.8 M Tris-HCl (pH 8.0). The inactivation of the Mn-S-state complex proceeded by apparent first-order kinetics, the rate constant dependent on NH2OH (CH3NHON) concentration and pH. In the range of micromolar concentrations of NH2OH, this inactivation did not occur via a cooperative type mechanism. Depletion of the 17 and 23 kDa proteins modified the pH dependency of inactivation (from pH 7.8 to 6.5) and also resulted in an approx. 2-fold maximum increase in the inactivation rate constant. Significantly, reconstitution of such NaCl-TMF-2 membranes with the 17 and 23 kDa proteins reverted both the pH dependency and the inactivation rate constant to that of TMF-2. A hierarchy of effectivity for solubilization of Mn and protein, which was highly correlated with inactivation of the Mn-S-state enzyme, was observed among NH2OH and its derivatives. This same hierarchy was observed irrespective of prior depletion of the 17 and 23 or the 17, 23 and 33 kDa proteins from TMF-2. The hierarchy of effectivity among derivatives was: NH2OH > CH3NHOH > NH2NH2, NH2OSO3 > NH2OCH3 ⪢ CH3NHOCH3, NH4Cl. The function(s) of the extrinsic PS II proteins as determinants of the reactivity of the Mn-S-state complex with polar amine vs other type compounds is discussed.

Requirements for the photoligation of Mn2+ in PS II membranes and the expression of water-oxidizing activity of the polynuclear Mn-catalyst

Ligation of Mn2+ into the polynuclear Mn‐catalyst of water oxidation was shown using PS II membranes depleted of their Mn and the 17, 23 and 33 kDa extrinsic proteins. This process specifically required light and Ca2+ concentrations of 5̃0 mM. Evidence was obtained indicating Mn2+/Ca2+ competition for Ca2+ and Mn2+ binding sites essential for the photoligation of Mn. Photoligation of Mn did not result in an increase of water oxidation capacity; however, water oxidation capacity was expressed following dark reconstitution minimally with the 33 kDa protein. The results represent the first observation of photoactivation of water oxidation in a system that excludes simple light‐driven Mn2+ transport across membrane(s).

Properties of a particulate nitrate reductase from the nodules of the soybean plant.

Abstract A particulate fraction has been isolated from Rhizobium japonicum cells of soybean nodules which catalyzes the reduction of nitrate to nitrite with either DPNH or succinate. The properties of the complex have been studied with each of these electron donors. When succinate is used as an electron donor for the nitrate reductase complex, the system exhibits many of the properties of succinic dehydrogenase and succin-oxidase which have been reported. These include competitive inhibition by malonate and pyrophosphate, inhibition by fluoride and an activation by phosphate. The nitrate reductase complex, with either succinate or DPNH as the electron donor was inhibited by Antimycin A, dicumarol, DNP and p -chloromercuribenzoate. Inhibition by the latter compound was prevented by either glutathione or cysteine. No evidence has been obtained for the involvement of a flavin in electron transport from either DPNH or succinate to nitrate; however, it is concluded that the failure to demonstrate a flavin requirement is associated with the difficulties in dealing with the particulate system. From studies of the inhibition of the nodule nitrate reductase by metal chelating agents and from kinetic studies of the cyanide inactivation of the system it is concluded that metal ions are involved in electron transport at two cyanide-sensitive sites with either DPNH or succinate as the electron donor. When succinate is used as the electron donor for the complex it is suggested that succinic dehydrogenase, a cytochrome, an Antimycin A-sensitive site, two cyanide sensitive sites, and nitrate reductase are involved in the electron transport to nitrate. When DPNH is used as a source of electrons for the system, the evidence indicates involvement of a vitamin K, or a related quinone, a cytochrome, an Anti-mycin A-sensitive site, two cyanide-sensitive sites, and nitrate reductase.

Depletion of Photosystem II-extrinsic proteins: II. Analysis of the PS II/water-oxidizing complex by measurements of N,N,N′,N′-tetramethyl-p-phenylenediamine oxidation following an actinic flash

Abstract The effects of selective extractions of Photosystem II (PS II) extrinsic proteins (with and without extraction of PS II Mn) on the coupling between the PS II trap and the S-state complex were determined by analysis of steady-state O 2 evolution, chemical reactivity of PS II Mn with N , N , N ′, N ′-tetramethyl- p -phenylenediamine (TMPD), and the kinetics of TMPD oxidation after a single short actinic flash. Unextracted PS II membranes showed only a slow approx. 60 ms component of TMPD oxidation (S 2 + TMPD → S 1 + TMPD + ), yielding an estimate of a PS II unit of 200–230 chlorophylls. Partial and complete inactivation of V O 2 by Tris or NH 2 OH gave a decrease of the S 2 component and a complementary increase of a fast approx. 600 μs component of TMPD oxidation (Z + + TMPD → Z + TMPD + ). CaCl 2 extraction of the 17, 23 and 33 kDa proteins, without solubilization of Mn from PS II membranes, made the fast component predominant, indicating most (up to 80%) of the traps were disconnected from the S-states; however, such extraction also made the Mn susceptible to rapid solubilization by TMPD. Extraction of only the 17 and 23 kDa proteins (NaCl-TMF-2) caused approx. 25% disconnection of the S-state complex, approx. 50% loss of V O 2 and an increase of charge loss reactions. Cl − depletion of NaCl-TMF-2 caused an increased extent of disconnection of the S-state complex and made PS II Mn susceptible to solubilization by TMPD. Reconstitution of NaCl-TMF-2 with the 17 and 23 kDa proteins abolished the approx. 25% disconnection and increased V O 2 , but did not abolish the charge loss path attributed to component C (Radmer, R., Cammarata, K., Tamura, N., Ollinger, O. and Cheniae, G. (1986) Biochim. Biophys. Acta 850, 21-32).

Photoactivation of NH2OH-treated leaves: reassembly of released extrinsic PS II polypeptides and religation of Mn into the polynuclear Mn catalyst of water oxidation

Inactivation of the water‐oxidizing enzyme by exposure of leaf segments to hydroxylamine results in a disassembly/perturbation of photosystem (PS) II extrinsic polypeptides (17>23 kDa) and solubilization of the tetra‐Mn complex. Religation of the tetra‐Mn complex, reassembly of these PS II extrinsic polypeptides with thylakoid membranes and reappearance of active water‐oxidizing activity all require light, and all are unaffected by inhibitors of protein synthesis and photophosphorylation. The results indicate, however, that the reassembly of these PS II extrinsic polypeptides into the multimeric water oxidizing enzyme occurs more rapidly than the photoligation of Mn as the tetra‐Mn complex.

Depletion of Photosystem II-extrinsic proteins. I. Effects on O2- and N2-flash yields and steady-state O2 evolution

Abstract Wheat O 2 -evolving Photosystem II (PS II) membranes having a PS II unit of approx. 200 chlorophylls (Chl), approx. 4 Mn/200 Chl, less than 1 P-700/3000 Chl and an electron-acceptor pool of approx. 2.5 equiv./PS II were analyzed and compared with wheat PS II membranes depleted (at least 90%) of the 17 and 23 kDa proteins by NaCl extraction during Triton X-100 isolation of membranes. Extraction of these proteins caused approx. 50% decrease in O 2 evolution in any light regime and an increase of approx. 2 equiv./PS II of the electron-acceptor pool, but affected neither Mn abundance, photoreduction of DCIP by tetraphenylboron, or N 2 yield (from NH 2 OH) from a single flash. Mass spectrometric analyses of O 2 flash yields in the presence of potassium ferricyanide showed that both chloroplasts and the unextracted PS II membranes yielded oscillations compatible with S 0 /S 1 /S 2 /S 3 of 25:75:0:0 and α (0.1) and β (0.05). Depletion of 17 and 23 kDa proteins resulted in a two-fold increase in α, approx. 25–40% disconnection of the S state complex from the PS II trap complex but with no change in β. Preincubation of control or extracted PS II membranes with potassium ferricyanide permitted a significant double-hit on the first flash. In the absence of an added electron acceptor, N 2 flash yields were more sustained with 17 and 23 kDa depleted than with 17 and 23 kDa sufficient PS II membranes. In contrast, no significant O 2 flash yields were observed with extracted PS II preparations under these conditions (control PS II membranes showed a predictable O 2 pattern before damping after only 5–6 flashes). These results suggest that extraction of the 17 and 23 kDa proteins results in an increase of pool size on the PS II acceptor side (seen as unmasking ‘Component C’). ‘Component C’ can mediate electron transfer from Q − to Z + (S 2 ).

A recollection of the development of the Kok-Joliot model for photosynthetic oxygen evolution

Twenty-five years of period-four O2-flash yield oscillation are celebrated with a personal recollection of the development of the Kok-Joliot model for photosynthetic oxygen evolution.

Photoinhibition of PSII Secondary Donors: Relevance to Photoactivation and Sites of Electron Donation

In the absence of either a functional water-oxidizing complex or artificial PSII electron donors, the secondary donors to P680 + are susceptible to weak-light photoinhibition by a mechanism not yet established [1–3]. The ability to restore O2 evolution through the photoactivation process is also weak-light sensitive [4]. We have studied these phenomena in attempts to identify factors which prohibit complete photoactivation, to determine if D+ is specifically required in this process, and to gain some insights into the specificity of Z+ and D+ for the photooxidation of exogenous PSII donors.