A.San Pietro

Evidence for the role of sulfhydryl groups in a pH-dependent transition of ferredoxin:NADP oxidoreductase

Abstract The pH profile of diaphorase activity of ferredoxin:NADP oxidoreductase suggests a pH-dependent transition between two forms of the enzyme. The apparent pH for this transition is about 8.0. The high-activity form, measured at pH 9.0, is strongly inhibited by increased salt concentration, whereas the low-activity form, measured at pH 7.0, is only weakly inhibited. Likewise, treatment of the enzyme with N-ethylmaleimide (NEM) inhibits the high-activity form more severely than the low-activity form. The data presented suggest that either high salt or NEM treatment converts the enzyme into a low-activity, pH-independent form. The data indicate that an ionized sulfhydryl group may be necessary for the high-activity form. With the exception of the ferredoxin-cytochrome c acceptor system, all other electron acceptors showed a pH dependence similar to that of the diaphorase activity.

Interaction of plastocyanin and P700 in PSI reaction center particles from C. reinhardi and spinach

Abstract Highly resolved Photosystem I (PSI) reaction center particles, solubilized from spinach and C. reinhardi chloroplasts with Triton X-100, catalyze very poorly the reduction of P700 by ascorbate at mildly acidic to alkaline pHs (pH 5–8). (Photooxidation of P700 means conversion of P700 to P700+; dark reduction of P700 is the reverse reaction.) The interaction between plastocyanin (PC) and P700 is assessed by measuring the stimulation of the rate of P700 reduction upon addition of PC at submicromolar concentrations. At a ratio of 2.6 PC per P700, about 20- to 25-fold stimulation of the rate of P700 reduction was observed in the presence of MgCl2 or CaCl2. Efficient interaction between PC and P700 occurred at near neutral pH (optimal pH = 6.6–6.8) and exhibited a preferential requirement for divalent metal cations. Monovalent cations are less effective in promoting transfer of electrons from PC to P700. At alkaline pHs (greater than 8.5) an accelerated rate of the PC-independent reduction of P700 was observed. Further, the PC-independent reaction at pH 9.4 showed a strong requirement for divalent cations. In contrast, monovalent cations are only partially effective in promoting the PC-independent reduction of P700 by ascorbate.

Interactions between spinach ferredoxin and other electron carriers. The involvement of a ferredoxin:cytochrome c complex in the ferredoxin-linked cytochrome c reductase activity of ferredoxin:NADP+ oxidoreductase.

Abstract The trinitrophenylation of a single amino group of spinach ferredoxin abolishes its ability to inhibit the diaphorase activity of the flavoprotein, ferredoxin:NADP oxidoreductase (EC 1.6.7.1); in contrast, the ability of ferredoxin to participate in the ferredoxin-linked cytochrome c reductase activity catalyzed by the flavoprotein is unaffected. Comparison with previously published results [Davis, D. J., and San Pietro, A. (1977) Biochem. Biophys. Res. Commun. 74 , 33–40]indicates that the site of interaction between ferredoxin and the flavoprotein resulting in inhibition if diaphorase activity is responsible for the spectrally observable 1:1 complex between the two proteins and is identical to the site of ferredoxin involvement in NADP photoreduction. The role of ferredoxin in the ferredoxin-linked cytochrome c reductase activity of the flavoprotein has been reexamined under conditions were the entire electron-accepting system (rather than just the ferredoxin component) is rate limiting. The data support a mechanism by which ferredoxin can bind either to the flavoprotein or to cytochrome c , and the ferredoxin:cytochrome c complex serves as the true substrate for reduction by the flavoprotein. Furthermore, Chromatographic evidence is presented for the formation of complexes between ferredoxin and cytochrome c .

Effect of uncouplers on anaerobic adaptation of hydrogenase activity in C reinhardtii.

Abstract An anaerobic incubation period of varying duration is required to induce hydrogenase activity in C. , reinhardtii . Inclusion of sodium acetate, a metabolizable carbonaceous substrate, in the medium during anaerobic incubation accelerates the activation process. Thus, in the presence of sodium acetate, hydrogen photoproduction is detected within 7 to 15 minutes after the onset of anaerobiosis. On the contrary, if an uncoupler of phosphorylation, such as CCCP or sodium arsenate, is present during anaerobic incubation, little activation of the hydrogenase is observed even after hours of anaerobic adaptation. Since the uncouplers had no inhibitory effect on hydrogen photoproduction by the alga when added to previously activated cells, they are not inhibitors of activated hydrogenase. The uncouplers interfere, most likely, with the activation of hydrogenase. Similar effects of uncouplers on the hydrogenase activation process were obtained using a cell-free assay of hydrogenase activity. These observations provide strong evidence that anaerobic activation of the hydrogenase is an energy requiring process.

On the reactivity of oxygen with photosystem I electron acceptors

The reduction of molecular oxygen as a consequence of photosynthetic electron transport was initially demonstrated [ 1,2] where oxygen was shown to serve as a Hill oxidant. Subsequently, it was recognized that the major site for reduction of oxygen is via photosystem I 13-51. In the absence of an artificial electron donor system, oxygen reduction by illuminated broken chloroplasts (poisoned with KCN to inhibit catalase) was shown [S] to be saturated by an oxygen tension equivalent to 4% (partial pressure) oxygen in nitrogen. Further, an app. Km of 5 FM was measured [S] for oxygen with the broken chloroplast preparation. Since soluble ferredoxin was assumed to be absent, the app. Km was suggested [S] to reflect the affinity of the PSI reductant (other than ferredoxin) for molecular oxygen. There is now substantial evidence to indicate that the endogenous Mehler reaction (oxygen reduction to produce hydrogen peroxide) involves a univalent reduction of oxygen and formation of a superoxide free radical [6,7]. The photoreduction of cytochrome c mediated by both ferredoxin and by superoxide radical was studied [7]. Photoreduction of cytoc~ome c, under aerobic conditions and in the presence of 0.5-I I.IM

Studies on photosystem I: I. Relationship of plastocyanin, cytochrome f and P700

Abstract Sonication of osmotically shocked chloroplasts in dilute pyridine yields a subchloroplast particle which is completely depleted of plastocyanin. Using dual-wave-length spectroscopy to study the light-induced spectral changes associated with these particles, photooxidation of reduced cytochrome f is not observed in the absence of added plastocyanin. The addition of plastocyanin, however, gives rise to a light-induced spectral change which is due solely to the oxidation of reduced cytochrome f and maximal response occurs in the presence of an equimolar amount of plastocyanin. These results are explained most simply by a strict linear sequence of electron transfer on the oxidizing side of Photosystem I from cytochrome f → plastocyanin → P700. It is found further that the presence of a detergent is required to elicit the light-induced cytochrome f oxidation signal, and that plastocyanin and the subchloroplast particles can be reconstituted by sonication. The reconstituted complex appears to be quite specific; showing a dissociation constant, during sonication, of 4.5 × 10 −7 m . This finding supports the theory that plastocyanin, in situ , is bound to the inner surface of the chloroplast membrane.

Studies on photosystem I: II. Involvement of ferredoxin in cyclic electron flow

Abstract The effects of ferredoxin (Fd) and ferredoxin-NADP reductase on the light-induced spectral changes of cytochrome f (cyt f ) were investigated with specific reference to their possible involvement in the cyclic electron transfort pathway of photosystem I (PS I). The steady-state level of photooxidation of reduced cytochrome f is decreased by ferredoxin but unaffected by either ferredoxin-NADP reductase alone or ferredoxin plus ferredoxin-NADP reductase when present in equimolar concentrations. These data are taken as evidence for a cyclic electron transport pathway of: PS I → “X” → Fd → (cyt f ) → PC → PS I. The reduced ferredoxin could either reduce directly plastocyanin (PC) or via cytochrome f ; the data do not allow differentiation between these two possibilities. However, neither ferredoxin-NADP reductase nor cytochrome b 564 appear to serve as electron carriers in this pathway.

Resolution and reconstitution of spinach ferredoxin-NADP+ reductase

Abstract The apoprotein from spinach ferredoxin-NADP + reductase was prepared by treatment with 3 M calcium chloride. This procedure caused complete removal of the FAD prosthetic group together with considerable denaturation of the apoprotein. Thus, the recovery of total activity upon reconstitution with FAD was only 30%. More importantly, however, both transhydrogenase and diaphorase activities were 70% of that native enzyme based on bound flavin. The visible spectrum and properties of the reconstituted reductase were undiscernible from those of the native protein.

Studies on photosystem I. Characteristics of "310 material" isolated from spinach chloroplasts.

Abstract Exposure of osmotically shocked chloroplasts to dilute pyridine and sonic oscillation results in the extraction of a small molecular-weight factor. Purification of the factor was accomplished using gel filtration chromatography. Due to the spectral nature of the purified species ( λ max at 310 nm) the factor was named “310 material.” Physiologically, the 310 material was found to inhibit a variety of ferredoxin-dependent photoreductions catalyzed by isolated spinach chloroplasts but stimulate both pseudocyclic photophosporylation and the ferredoxin-independent photoreduction of mammalian cytochrome c . The latter reaction was found to involve, at least partially, the formation of a Superoxide radical. Dark-reduction studies have further established that the 310 material is an autooxidizable electron carrier. Chemically, the 310 material is a water-soluble, low molecular-weight phenolic-type compound; possibly a derivative of coumaric acid. No proteinaceous material is observed in physiologically active preparations of 310 material. Based on these findings, it is concluded that the isolated 310 material acts on the reducing side of Photosystem I at or near the site of reduction of ferredoxin and competes with ferredoxin for the reducing power generated by the Photosystem I reaction center. The exact physiological role of the 310 material in the intact photosynthetic system, however, remains unknown. The similarities between the 310 material and a variety of other factors previously isolated from chloroplasts are discussed.

Effect of oxygen and pentachlorophenol on the induction lag of photosynthesis in Chlamydomonas reinhardi.

Abstract Following dark anaerobic incubation Chlamydomonas reinhardi exhibit a pronounced induction lag in photosynthesis; in contrast, dark aerobic incubation caused only a minimal induction lag. Addition of a low concentration of the uncoupler, pentachlorophenol (PCP), to respiring cells in darkness extended the induction period. Far red light preillumination partially overcame the induction lag resulting from dark anaerobic treatment or uncoupling with PCP in aerobic medium. The induction lag in photosynthesis is explained in terms of intracellular energy reserve and its effects on the level of phosphorylated intermediates of the Calvin cycle e.g., RuDP, in the dark. Further, evidence is presented which demonstrates that: (a) PCP, at concentrations below 10 μ m , preferentially uncouples oxidative phosphorylation in vivo and (b) photophosphorylation, both in in vivo and in vitro , is much more resistant.