Influence of some metal concentrations on the activity of antioxidant enzymes and concentrations of vitamin E and SH-groups in the digestive gland and gills of the freshwater bivalve Unio tumidus from the Serbian part of Sava River

We examined whether the freshwater bivalve Unio tumidus from the Sava River can serve as a bioindicator organism for long-term biomonitoring of river ecosystems for the presence of metal pollutants. To this end, we assessed in the digestive glands and gills of mussels, changes in activity of antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR), the phase II biotransformation enzyme glutathione-S-transferase (GST)), and changes in the concentrations of the non-enzymatic components of the antioxidant system (vitamin E and sulfhydryl groups (-SH), after exposure to metals in the environment. Mussels were collected at four sites where the concentrations of dissolved metals (Cu, Cd, Zn, Fe, Mn, Hg, Ni, As, Pb) were quantified. Cu, Ni and As exerted concentration-dependent inhibitory effects on CAT and GST activities. Increasing concentrations of Cd promoted increases in GSH-Px activity and -SH concentration. In response to increased Zn concentration GR activity increased whereas Fe promoted decreased enzymatic activity. Negative correlations between the concentrations of Cu and Cd and vitamin E, and a positive correlation between Mn and vitamin E concentrations were detected. The described correlations between components of the antioxidant system and metal levels in the environment reveal a high physiological sensitivity of freshwater mussels to pollution, supporting their use in biomonitoring of metal contamination in river ecosystems.     

Metallothionein concentration in sponges (Spongia officinalis) as a biomarker of metal contamination

The synthesis of metallothioneins (MTs) is often induced when organisms are exposed to heavy metals in the field. They are among the major "specific" biomarkers identified to date. With a view to include MTs in biomonitoring programs, the organisms most commonly studied are bivalves. Sponges present most of the characteristics researched in bioindicators of pollution and consequently have been proposed to constitute a "Sponge Watch Program". The detection of large quantities of metals in sponges suggests the existence of detoxification systems and indeed, the presence of metallothionein-like proteins (MTLPs) has been reported in two different species of sponges. In Spongia officinalis, the present study has demonstrated the presence of compounds exhibiting most of the characteristics of MTs: cytosolic, heat-stable, with apparent molecular mass of 4 to 15 kDa and binding (at least) Ag, Cu and Zn. Specimens have been collected along the French Mediterranean coast from three sites differing by their degree of contamination. Relationships between MTLP and metal concentrations have been established. For copper, mercury and zinc, the correlations were significantly positive.     

Differential pulse polarography: a method of directly measuring uptake of metal ions by live bacteria without separation of biomass and medium

Abstract The technique of differential pulse polarography is shown here for the first time to be applicable to monitoring directly the uptake of metal ions from solution by live bacteria in the chamber of the polarograph. The potential at which the polarographic current peak is observed is characteristic of the metal, whereas peak height is proportional to metal concentration. Adding solutions of Cd(II) or Zn(II) to a suspension of Pseudomonas cepacia in 50 mM Hepes buffer (pH 7.4) in the chamber gave polarographic peaks of lower amplitude than those observed when these metal solutions were added to buffer alone, due to metal binding or uptake by cells. Langmuir plots gave binding capacities of 0.13 and 0.20 mmol metal (Dd or Zn, respectively) per g (dry weight) biomass. Ni(II) uptake was biphasic. Metal uptake increased with pH. The value of polarography for rapid assessment of metal removal by cells and the ability to measure uptake from multi-metal solutions is demonstrated.     

Comparison of metallothionein concentrations and tissue distribution of trace metals in crabs (Pachygrapsus marmoratus) from a metal-rich estuary,in and out of the reproductive season

Crabs, Pachygrapsus marmoratus, were sampled in June 1997 and February 1998 from two sites (at the mouth and 25 km upstream) in the metal-rich Gironde estuary, France. Gills and hepatopancreas were analysed for metal (Cd, Cu, Zn) and metallothionein (MT) contents, in order to examine the influence of both biological and environmental factors on the physico-chemical forms of detoxified metal storage in the crabs. The concentrations of MT and both cytosolic and insoluble metals were not greatly different between males and females, and the influence of organ weights was also minimal. Intersite differences were observed, probably resulting from the gradient of salinity in the estuary, which interacts with both the chemical speciation and bioavailability of metals, and the general protein metabolism of the crabs. Seasonal changes were also important, probably in interaction with the moult and reproductive cycles. In February, concentrations of insoluble metals were generally higher than in June, in both organs, suggesting that essential metals, particularly Zn, are stored during winter then remobilised during the breeding season. The natural variability in the concentrations of MT often concealed any relationship with accumulated metal concentrations. Thus MT in crabs cannot be considered as a useful biomarker of metal pollution.     

Effects of ionic and complexed metal concentrations on plant uptake of cadmium and micronutrient metals from solution

Summary Uptake of Cd and micronutrient metals by intact tomato plants (Lycopersicon esculentum, cv. Wisconsin-55) from solution cultures was investigated by establishing four levels of Cd-ion activity in the presence or absence of a metal-complexing agent (±EDTA). Activity ratios of Cd, Cu, Mn, Ni, and Zn were controlled with chelating resin while activity ratios of K, Ca, and Mg were controlled with a strong-acid cation-exchange resin. Hydrogen ion activity was controlled with a weak-acid cation-exchange resin and P activity by a cation-exchange resin containing adsorbed polynuclear hydroxy-Al. The concentrations of all nutrients and Cd were maintained at concentrations similar to those occuring in solutions of sludge-amended soils. The EDTA treatments increased the concentrations of Cu and Ni in hydroponic solution by approximately four orders of magnitude, Zn by two orders of magnitude, Cd by a factor of 50, Mn by a factor of 2.4, and Fe by a factor of 1.6 Neither the Cd nor the EDTA treatments affected plant yield, and Cd treatments did not significantly affect uptake of other elements. EDTA treatments inhibited Fe uptake, enhanced Cu uptake, and had little effect on the uptake of Cd, Zn, and Mn. Accumulation of Cd, Zn, Mn, and Cu in plant shoots appears to be related to their respective ionic activities rather than their concentrations in hydroponic solution. Research supported by the College of Agricultural and Life Sciences, University of Wisconsin-Madison and by the United States Environmental Protection Agency through Grant CR807270010.     

A method for controlling the activities of free metal,hydrogen, and phosphate ions in hydroponic solutions using ion exchange and chelating resins

Summary An ion exchange and chelating resin system was developed to buffer the activities of selected free cations and phosphate in hydroponic solutions at concentrations similar to those that occur naturally in soil solutions. Free-ion activities of Cd, Cu, Ni, Zn, Mn, H, Ca, Mg, and K were maintained by ion exchange and chelating resins in a controlled ionic strength system. Iron was controlled by EDDHA and chelating resin, and P by a cation-exchange resin containing adsorbed polynuclear hydroxyaluminum. This mixed-resin hydroponic system was used to establish a range of ionic Cd activities similar to that found in soil solutions of soils amended with sewage sludge. Activities of other nutrients were maintained at realistic soil-solution levels. A metal complexing agent (EDTA) was used to increase total metal concentration in the hydroponic solutions without significantly altering the ionic activities of the metals maintained in solution. This allowed differentiation of the effects of free ions and complexed metals on metal uptake. Concentrations of metal complexes in solution were controlled by the ion activities of the metals maintained and the concentrations and selectivities of the complexing agent. The mixed-resin system supplied sufficient nutrients for the growth of tomato plants (Lycopersicon esculentum, cv. Wisconsin-55) in hydroponic culture. Research supported by the College of Agricultural and Life Sciences, University of Wisonsin-Madison and by United States Environmental Protection Agency through Grant CR807270010.     

Effects of copper,zinc, and cadmium ions on the production of phosphate from phytic acid by the phytase system in spruce forest soil

Summary The hydrolysis of phosphate from phytic acid by the acid soil phytase system was reduced in the presence of metal ions. Copper was most effective in this respect — zinc and cadmium were less inhibitory. Binding to metals did not completely inhibit the hydrolysis of phytic acid. At higher metal concentrations, where binding to other soil constituents, like humic acids, interfered less, the inhibition of the phytase activity was stronger than that of acid phosphatase.     

Differential effects of AM fungal isolates on Medicago truncatula growth and metal uptake in a multimetallic (Cd, Zn, Pb) contaminated agricultural soil

Toxic metal accumulation in soils of agricultural interest is a serious problem needing more attention, and investigations on soil–plant metal transfer must be pursued to better understand the processes involved in metal uptake. Arbuscular mycorrhizal (AM) fungi are known to influence metal transfer in plants by increasing plant biomass and reducing metal toxicity to plants even if diverging results were reported. The effects of five AM fungi isolated from metal contaminated or non-contaminated soils on metal (Cd, Zn) uptake by plant and transfer to leachates was assessed with Medicago truncatula grown in a multimetallic contaminated agricultural soil. Fungi isolated from metal-contaminated soils were more effective to reduce shoot Cd concentration. Metal uptake capacity differed between AM fungi and depended on the origin of the isolate. Not only fungal tolerance and ability to reduce metal concentrations in plant but also interactions with rhizobacteria affected heavy metal transfer and plant growth. Indeed, thanks to association with nodulating rhizobacteria, one Glomus intraradices inoculum increased particularly plant biomass which allowed exporting twofold more Cd and Zn in shoots as compared to non-mycorrhizal treatment. Cd concentrations in leachates were variable among fungal treatments, but can be significantly influenced by AM inoculation. The differential strategies of AM fungal colonisation in metal stress conditions are also discussed.     

Metal ion-catalysed hydrolysis of ampicillin in microbiological growth media

Anodic stripping voltammetry of bacterial growth medium containing copper(II) and ampicillin shows that Cu(II) is complexed by the antibiotic and that this complex decomposes to give Cu(II) complexes with ligands derived from ampicillin. At pH 7, substantial decomposition of ampicillin occurs over a few minutes, and even the very low levels of Cu(II) in Chelex-extracted medium are able effectively to catalyse the decomposition. The significance of this observation was shown during the screening of an Escherichia coli cosmid library for clones exhibiting increased resistance to Zn(II), Co(II) or Cd(II); the unexpected growth of the ampicillin-sensitive host E. coli strain on Luria-Bertani plates containing ampicillin and any of these metals was attributed to metal ion-catalysed decomposition of ampicillin. The instability of ampicillin (and other beta-lactam antibiotics) to metal ion-catalysed hydrolysis means that great care must be taken to ensure that such reactions do not occur in growth media. Furthermore, it is clear that double selection for resistance to ampicillin and metals such as Cu(II), Zn(II), Co(II) and Cd(II) is impossible.     

Archaeal and bacterial communities of heavy metal contaminated acidic waters from zinc mine residues in Sepetiba Bay

Mining of metallic sulfide ore produces acidic water with high metal concentrations that have harmful consequences for aquatic life. To understand the composition and structure of microbial communities in acid mine drainage (AMD) waters associated with Zn mine tailings, molecular diversity of 16S genes was examined using a PCR, cloning, and sequencing approach. A total of 78 operational taxonomic units (OTUs) were obtained from samples collected at five different sites in and around mining residues in Sepetiba Bay, Brazil. We analyzed metal concentration, physical, chemical, and microbiological parameters related to prokaryotic diversity in low metal impacted compared to highly polluted environments with Zn at level of gram per liter and Cd–Pb at level of microgram per liter. Application of molecular methods for community structure analyses showed that Archaea and Bacteria groups present a phylogenetic relationship with uncultured environmental organisms. Phylogenetic analysis revealed that bacteria present at the five sites fell into seven known divisions, α-Proteobacteria (13.4%), β-Proteobacteria (16.3%), γ-Proteobacteria (4.3%), Sphingobacteriales (4.3%), Actinobacteria (3.2%) Acidobacteria (2.1%), Cyanobacteria (11.9%), and unclassified bacteria (44.5%). Almost all archaeal clones were related to uncultivated Crenarchaeota species, which were shared between high impacted and low impacted waters. Rarefaction curves showed that bacterial groups are more diverse than archaeal groups while the overall prokaryotic biodiversity is lower in high metal impacted environments than in less polluted habitats. Knowledge of this microbial community structure will help in understanding prokaryotic diversity, biogeography, and the role of microorganisms in zinc smelting AMD generation and perhaps it may be exploited for environmental remediation procedures in this area.     

厦门海域两头中华白海豚体内微量元素的积累

对2004年4月在厦门海域搁浅死亡的两头中华白海豚（Sousa chinensis）进行了肝脏、胰、胃、肾、肠、肺、肌肉、心脏等多组织微量元素锌（Zn）、铜（Cu）、汞（Hg）、铅（Pb）、镉（Cd）和砷（As）浓度的测定。两豚各组织总体上必需元素Zn、Cu浓度高于有毒元素Hg、Pb、Cd和As。XM20040430体内特别高的Zn、Hg、Pb、Cd浓度分别出现于皮肤（500μg/g干重）、肝脏（255μg/g干重）、肺（13.0μg/g干重）和肾（2.82μg/g干重）中,而Cu和As在各组织中的浓度较为相似。XM20040429体内仅肝脏中Zn（448μg/g干重）和Cu（52.0μg/g干重）的浓度远高于其他组织。虽然两海豚微量元素的总体积累多低于急性毒性水平,但有毒元素Hg、Pb、Cd、As的明显检出和XM20040429肝脏中异常高的Zn浓度均显示出进一步研究微量元素慢性胁迫效应的必要性。结果还显示厦门港海域近年来Hg、Cd、Pb污染未见减轻,As也明显检出,必须引起高度重视。今后有关水生哺乳动物中As积累的研究也有待加强。     

Trace metal concentrations in two New Zealand talitrid amphipods: effects of gender and reproductive state and implications for biomonitoring

This is the first in-depth investigation of whether the gender or reproductive state of talitrid amphipods affects the bioaccumulation of trace metals. Concentrations of copper, zinc, and cadmium were measured in the beach flea Transorchestia chiliensis (Milne-Edwards) and the sand hopper Talorchestia quoyana (Milne-Edwards) (Amphipoda: Talitridae) from sites in and near the Avon-Heathcote Estuary, Christchurch, New Zealand. For T. chiliensis, the whole body trace metals concentrations (μg g⁻¹) were generally similar for nonbrooding, brooding, and brooding females that had the embryos removed. Where there were differences between female groups (3 out of 15 samples), concentrations in nonbrooding females were below those for brooding females. The trace metal concentrations of separated embryos did not follow those of their mothers. The body zinc concentration was similar for males and females. For copper and cadmium, body concentrations for females were higher than males at the two most contaminated sites. Cadmium body concentrations were similar between sites, and the lowest concentrations were from amphipods from one of the Estuary sites rather than the reference site. In T. quoyana, the trace metal concentrations in nonbrooding female and male sand hoppers were similar for copper and zinc, but cadmium concentrations were higher in nonbrooding females than in males. Copper and zinc concentrations within amphipod body tissues did not reflect those in the sediment or their food. The implications of these results are discussed in relation to previous studies and the use of beach fleas and sand hoppers as metal biomonitors. The beach flea T. chiliensis is recommended as a suitable trace metal biomonitor in New Zealand coastal waters with the potential to be affected by anthropogenic trace metal contamination.     

Effects of a phosphorus amendment and the pH of water used for watering on the mobility and phytoavailability of Cd, Pb and Zn in highly contaminated kitchen garden soils

Studies on two lead and zinc smelters in Northern France (Metaleurop Nord and Umicore) showed that the level of metallic contamination of kitchen garden soils is higher than the agricultural soils located in the same environment. This results most particularly from cropping practices and the addition of various products. Due to the physical and chemical parameters of these soils, the behaviour and transfer of pollutants towards various plants (grass, trees, and vegetables) may be perceptibly different than what is observed on agricultural soils.For a better understanding of pollutant behaviour in kitchen garden topsoils, the Cd, Pb and Zn was fractionated using the SM&T protocol and various extracting solutions (CaCl₂, acetic acid, and citric acid) to evaluate their mobility in two highly contaminated soils chosen in the area affected by the past atmospheric emissions of the two smelters. In addition, agricultural topsoil was sampled in a non-massively contaminated area and was therefore chosen as the control soil.The three soils were amended with a mixture of hydroxyapatite (HA) and diammonium phosphate (DAP). At 6 months, extracting procedures were carried out to evaluate the effects of the amendment on the mobility of Cd, Pb and Zn. This step was then supplemented by an evaluation of the impact of the amendment on the phytoavailability of pollutants, which was determined in plant uptake studies with ryegrass (Lolium perenne L.) by considering only the pollutant concentrations in their shoots. Two experiments were carried out. In the first one, unamended and amended soils and ryegrass were watered with distilled water (pH = 7). In the second one, osmosed water (pH = 5.5) was used to evaluate the effects of the acid water-phosphate amendment system on the mobility and phytoavailability of Cd, Pb and Zn. Six months after the start of the experiments, the selective extractions showed that the effectiveness of the amendment studied depended on the element, the soil and the water's pH. Reductions of metal eluted from the contaminated soils were 1.5-37.9% for Cd, and 9.1-80.9% for Pb. Application of P amendment to the combination of osmosed water was generally the most effective for immobilising Cd and Pb elution. In contrast, the mixture of HA and DAP was ineffective for reducing Zn elution. The plant-fresh biomass yield was significantly (p < 0.05) increased by the combination of P amendment and distilled water, whereas a reduction of biomass was recorded with the combined amendment and osmosed water. Addition of P amendment generally reduced Pb uptake in ryegrass shoots (1-47%), while both Cd and Zn were increased by 17.9-79% and 0.45-100%, respectively.     

The use of a white rot fungi (Pleurotus ostreatus) immobilized on Amberlite XAD-4 as a new biosorbent in trace metal determination

The present work proposes the use of Pleurotus ostreatus immobilized on Amberlite XAD-4 as new biosorbent in trace metal determination. The effects of experimental parameters, such as “pH and flow rate of sample solution, amount of solid phase, eluent type, and concentration” on the recovery of the metal ions were investigated. Maximum adsorption of Cr(III), Cd(II) and Cu(II) ions took place in the pH range 4-5. These metal ions can be desorbed with 1 M HCl (recovery 95-100%). 0.2 g adsorbent amount and 2.5 mL min⁻¹ flow rate was found to be optimum of all preconcentration experiments. The sorption capacity after 10 cycles of sorption and desorption does not vary more than 2.0%. The influences of the contaminant ions on the retentions of the analytes were also examined. The results showed that P. ostreatus immobilized on Amberlite XAD-4 can be considered as very promising material in trace metal determination.     

Temporal variations of metallothionein and metal concentrations in the digestive gland of oysters (Crassostrea gigas) from a clean and a metal-rich site

The concentrations of metallothionein (MT) in bivalves, a potential biomarker of metal pollution, are variable according to specific organs, the highest concentrations being encountered in the digestive glands of oysters. Thus, the present study has been focussed on this organ with a view to validate the use of MT as a biomarker in the field, the temporal changes of metal and metallothionein concentrations have been examined from March to October 1997 in the digestive gland of resident oysters from a clean site (Bay of Bourgneuf, France) and a metal-rich site, the Gironde estuary which has been shown as the most Cd-contaminated marine area in France but is also enriched with Cu and Zn. Moreover, oysters from the clean site have been translocated to the Gironde estuary over the same period. Taking into account all the samples collected over the 7 months of the study, MT concentrations in the digestive gland were positively correlated with weight whereas metal levels were negatively correlated with weight. However, considering monthly samples including specimens from both sites (resident or translocated oysters), a positive correlation was shown between MT and metal concentrations in autumn (September and October) but not in spring and summer. These findings limit the interest of using the digestive gland of oysters as the preferred tissue for the determination of MT concentration as a biomarker.The alternative use of gills should be considered.     

Kinetic properties of a micronutrient transporter from<Emphasis Type="Italic"> Pisum sativum</Emphasis> indicate a primary function in Fe uptake from the soil

Fe uptake in dicotyledonous plants is mediated by a root plasma membrane-bound ferric reductase that reduces extracellular Fe(III)-chelates, releasing Fe²⁺ ions, which are then absorbed via a metal ion transporter. We previously showed that Fe deficiency induces an increased capacity to absorb Fe and other micronutrient and heavy metals such as Zn²⁺ and Cd²⁺ into pea (Pisum sativum L.) roots [Cohen et al. (1998) Plant Physiol 116:1063–1072). To investigate the molecular basis for this phenomenon, an Fe-regulated transporter that is a homologue of the Arabidopsis IRT1 micronutrient transporter was isolated from pea seedlings. This cDNA clone, designated RIT1 for root iron transporter, encodes a 348 amino acid polypeptide with eight putative membrane-spanning domains that is induced under Fe deficiency and can functionally complement yeast mutants defective in high- and low-affinity Fe transport. Chelate buffer techniques were used to control Fe²⁺ in the uptake solution at nanomolar activities representative of those found in the rhizosphere, and radiotracer methodologies were employed to show that RIT1 is a very high-affinity ⁵⁹Fe²⁺ uptake system (K _m =54–93 nM). Additionally, radiotracer (⁶⁵Zn, ¹⁰⁹Cd) flux techniques were used to show that RIT can also mediate a lower affinity Zn and Cd influx (K _m of 4 and 100 M, for Zn²⁺ and Cd²⁺, respectively). These findings suggest that, in typical agricultural soils, RIT1 functions primarily as a high-affinity Fe²⁺ transporter that mediates root Fe acquisition. This is consistent with recent findings with Arabidopsis IRT1 knockout mutants that strongly suggest that this transporter plays a key role in root Fe uptake and nutrition. However, the ability of RIT1 to facilitate Zn and Cd uptake when these metals are present at elevated concentrations suggests that RIT1 may be one pathway for the entry of toxic metals into the food chain. Furthermore, the finding that plant Fe deficiency status may promote heavy metal uptake via increased expression of this transporter could have implications both for human nutrition and also for phytoremediation, the use of terrestrial plants to sequester toxic metals from contaminated soil.     

Variations in the coordination environment of Co, Cu and Zn complexes prepared from a tridentate (imino)pyridine ligand and their structural comparisons

The variations in the coordination environment of Co(II), Cu(II) and Zn(II) complexes with the neutral, tridentate ligand bis[1-(cyclohexylimino)ethyl]pyridine (BCIP) are reported. Analogous syntheses were carried out utilizing either the M(BF₄)₂ · xH₂O or MCl₂ · xH₂O metal salts (where M = Co(II), Cu(II) or Zn(II)) with one equivalent of BCIP. When the hydrated metal starting material was used, cationic, octahedral complexes of the type [M(BCIP)₂]²⁺ were isolated as the tetrafluoroborate salt (4, 5). Conversely, when the hydrated chloride metal salt was used as the starting material, only neutral, pentacoordinate [M(BCIP)Cl₂] complexes (1-3) formed. All complexes were characterized by X-ray diffraction studies. The three complexes that are five coordinate have distortions due mainly to the pyridine di-imine bite angle. The [Cu(BCIP)Cl₂] (2) also exhibits deviations in the Cu(II)-Cl bond distances with values of 2.4242(9) and 2.2505(9) Å, which are not seen in the analogous Zn(II) and Co(II) structures. Similarly, the two six coordinate complexes (5, 6) are also altered by the ligand frame bite angle giving rise to distorted octahedral geometries in each complex. The [Cu(BCIP)₂](BF₄)₂ (6) also exhibits Cu(II)-N_imine bond lengths that are on average 0.14 Å longer than those found in the analogous 5 coordinate complex, [Cu(BCIP)Cl₂]. In addition to X-ray analysis, all complexes were also characterized by UV/Vis and IR spectroscopy with ¹H NMR spectroscopy being used for the analysis of the Zn(II) analogue (3).     

Effect of NADP+ on light-induced cytochrome changes in membrane fragments from a blue-green alga

The effect of NADP⁺ on light-induced steady-state redox changes of membrane-bound cytochromes was investigated in membrane fragments prepared from the blue-green algae Nostoc muscorum (Strain 7119) that had high rates of electron transport from water to NADP⁺ and from an artificial electron donor, reduced dichlorophenolindophenol (DCIPH₂) to NADP⁺. The membrane fragments contained very little phycocyanin and had excellent optical properties for spectrophotometric assays. With DCIPH₂ as the electron donor, NADP⁺ had no effect on the light-induced redox changes of cytochromes: with or without NADP⁺, 715- or 664-nm illumination resulted mainly in the oxidation of cytochrome f and of other component(s) which may include a c-type cytochrome with an α peak at 549 nm. With 664 nm illumination and water as the electron donor, NADP⁺ had a pronounced effect on the redox state of cytochromes, causing a shift toward oxidation of a component with a peak at 549 nm (possibly a c-type cytochrome), cytochrome f, and particularly cytochrome b₅₅₉. Cytochrome b₅₅₉ appeared to be a component of the main noncyclic electron transport chain and was photooxidized at physiological temperatures by Photosystem II. This photooxidation was apparent only in the presence of a terminal acceptor (NADP⁺) for the electron flow from water.