Alice Haddy

EPR spectroscopy of the manganese cluster of photosystem II

Electron paramagnetic resonance (EPR) spectroscopy is a valuable tool for understanding the oxidation state and chemical environment of the Mn4Ca cluster of photosystem II. Since the discovery of the multiline signal from the S2 state, EPR spectroscopy has continued to reveal details about the catalytic center of oxygen evolution. At present EPR signals from nearly all of the S-states of the Mn4Ca cluster, as well as from modified and intermediate states, have been observed. This review article describes the various EPR signals obtained from the Mn4Ca cluster, including the metalloradical signals due to interaction of the cluster with a nearby organic radical.

Effects of azide on the S2 state EPR signals from Photosystem II

The anion azide, N3-, has been previously found to be an inhibitor of oxygen evolution by Photosystem II (PS II) of higher plants. With respect to chloride activation, azide acts primarily as a competitive inhibitor but uncompetitive inhibition also occurs [Haddy A, Hatchell JA, Kimel RA and Thomas R (1999) Biochemistry 38: 6104–6110]. In this study, the effects of azide on PS II-enriched thylakoid membranes were characterized by electron paramagnetic resonance (EPR) spectroscopy. Azide showed two distinguishable effects on the S2 state EPR signals. In the presence of chloride, which prevented competitive binding, azide suppressed the formation of the multiline and g = 4.1 signals concurrently, indicating that the normal S2 state was not reached. Signal suppression showed an azide concentration dependence that correlated with the fraction of PS II centers calculated to bind azide at the uncompetitive site, based on the previously determined inhibition constant. No evidence was found for an effect of azide on the Fe(II)QA- signals at the concentrations used. This result is consistent with placement of the uncompetitive site on the donor side of PS II as suggested in the previous study. In chloride-depleted PS II-enriched membranes azide and fluoride showed similar effects on the S2 state EPR signals, including a notable increase and narrowing of the g = 4.1 signal. Comparable effects of other anions have been described previously and apparently take place through the chloride-competitive site. The two azide binding sites described here correlate with the results of other studies of Lewis base inhibitors.

Transition metal and organic radical components of carp liver tissue observed by electron paramagnetic resonance spectroscopy

Abstract Paramagnetic transition metal centers and organic radicals in liver from wild-type carp ( Cyprinus carpio ) were characterized by electron paramagnetic resonance (EPR) spectroscopy. Approximately twelve EPR signals were observed at 77 K with resonance positions between g =1.8 and g =2.5. Identification was facilitated by a study of the variation in signal intensity with microwave power (microwave power saturation) for each signal. Many were organic radical or iron signals from typical liver enzymes, including cytochrome P450, coenzyme Q 10 , NADH dehydrogenase, and succinate dehydrogenase, cytochrome c oxidase and/or catalase. Of special interest were two signals that are not normally found in mammalian liver. The first was a six-line signal from divalent manganese, which was evident in the spectra in quantities suggestive of a functional role. The second was probably a signal from nitrosylated non-heme iron and may be related to the presence of nitrogen-containing compounds produced by nitrifying bacteria in the aquatic environment. These notable differences between the EPR spectra of fish and mammalian liver suggest major metabolic differences between the two systems.

An alternative method for calcium depletion of the oxygen evolving complex of photosystem II as revealed by the dark-stable multiline EPR signal.

The dark-stable multiline EPR signal of photosystem II (PSII) is associated with a slow-decaying S(2) state that is due to Ca(2+) loss from the oxygen evolving complex. Formation of the signal was observed in intact PSII in the presence of 100-250 mM NaCl at pH 5.5. Both moderately high NaCl concentration and decreased pH were required for its appearance in intact PSII. It was estimated that only a portion of oxygen evolving complexes was responsible for the signal (about 20% in 250 mM NaCl), based on the loss of the normal S(2)-state multiline signal. The formation of the dark-stable multiline signal in intact PSII at pH 5.5 could be reversed by addition of 15 mM Ca(2+) in the presence of moderately high NaCl, confirming that it was the absence of Ca(2+) that led to its appearance. Formation of the dark-stable multiline signal in NaCl-washed PSII, which lacks the PsbP (23 kDa) and PsbQ (17 kDa) subunits, was observed in about 80% of the sample in the presence of 150 mM NaCl at pH 5.5, but some signal was also observed under normal buffer conditions. In both intact and NaCl-washed PSII, the S(2)Y(Z). signal, which is also characteristic of Ca(2+) depletion, appeared upon subsequent illumination. Formation of the dark-stable multiline signal took place in the absence of Ca(2+) chelator or polycarboxylic acids, indicating that the signal did not require their direct binding as has been proposed previously. The conditions used here were milder than those used to produce the signal in previous studies and included a preillumination protocol to maximize the dark-stable S(2) state. Based on these conditions, it is suggested that Ca(2+) release occurred through protonation of key residues that coordinate Ca(2+) at low pH, followed by displacement of Ca(2+) with Na(+) by mass action at the moderately high NaCl concentration.

The Cl − Requirement for Oxygen Evolution by Photosystem II Explored Using Enzyme Kinetics and EPR Spectroscopy

Chloride is a well-known activator of oxygen evolution activity in photosystem II. Its effects have been characterized over several decades of research, as methods have developed and improved. By replacing chloride with other small anions with a range of chemical properties, a picture of the requirements of a successful anion activator can be formulated. In this review, the results of experiments on the chloride effect using enzyme kinetics methods and electron paramagnetic resonance spectroscopy are described, with summaries for the major anion activators and inhibitors that have been studied.

Characterization of fluoride inhibition in photosystem II lacking extrinsic PsbP and PsbQ subunits

Photosynthetic oxygen evolution occurs through the oxidation of water at a catalytic Mn4CaO5 cluster in photosystem II and is promoted by chloride, which binds at two sites near the Mn4CaO5 cluster. Fluoride is a competitive inhibitor of chloride activation, but study of its effects is complicated by the possibility that it may form an insoluble CaF2 complex. In this study, the effects of fluoride were studied using PSII lacking the PsbP and PsbQ subunits, which help to regulate the requirements for the inorganic cofactors Ca2+ and Cl-. In this preparation, which allows easy exchange of ions, it was found that F- does not directly remove Ca2+ even when catalytic turnovers take place, suggesting that fluoride is not able to access the inner coordination sphere of Ca2+. By monitoring the loss in O2 evolution activity, the dissociation constant of F- was estimated to be about 1 mM in intact PSII, consistent with previous studies, and about 77 mM in PSII lacking the extrinsic subunits. The significantly higher value for PSII lacking PsbP and PsbQ is consistent with results for other ions. The effects of F- on electron transfer to Tyr Z was also studied and found to show similar trends in PSII with and without the two extrinsic subunits, but with a more pronounced effect in PSII lacking the extrinsic subunits. These results indicate that in PSII lacking PsbP and PsbQ, fluoride does not directly interact with or remove Ca2+ and inhibits O2 evolution in a manner comparable to PSII with the extrinsic subunits intact.

Studies of Photosystem II Using Azide as an Inhibitor of Oxygen Evolution

Chloride is required for proper function of the oxygen evolving complex in Photosystem II (P SIT). Chloride-depletion and replacement of chloride with some anions, such as F−, interferes with normal formation of the EPR signals from the S2 state, suppressing the multiline signal and causing an increase in the g=4.1 signal (1, 2). Chloride binds to one high affinity, slow exchange site in PSII with dissociation constant Kd =20 μM (3). This site goes into a lower affinity form after chloride-depletion (3).