Non-functional State Research Articles

Hibernating Peripheral Nerve

AbstractIt is proposed that under certain circumstances peripheral nerve may exist in a poorly or nonfunctional reversible state [termed hibernating peripheral nerve (HPN)] after neural insult. Furthermore, it is speculated that certain high intraneural concentrations of lidocaine may conceivably facilitate a hypothetical state of hibernating peripheral nerve.

A Simple Molecular Model for Thermophilic Adaptation of Functional Nucleic Acids

RNA molecules have numerous functions including catalysis and small molecule recognition, which typically arise from a tertiary structure. There is increasing interest in mechanisms for the thermostability of functional RNA molecules. Sosnick, Pan, and co-workers introduced the notion of "functional stability" as the free energy of the tertiary (functional) state relative to the next most stable (nonfunctional) state. We investigated the extent to which secondary structure stability influences the functional stability of nucleic acids. Intramolecularly folding DNA triplexes containing alternating T*AT and C+*GC base triples were used as a three-state model for the folding of nucleic acids with functional tertiary structures. A four-base-pair tunable region was included adjacent to the triplex-forming portion of the helix to allow secondary structure strength to be modulated. The degree of folding cooperativity was controlled by pH, with high cooperativity maintained by lower pH (5.5), and no cooperativity by higher pH (7.0). We find a linear relationship between functional free energy and the free energy of the secondary structure element adjacent to tertiary interactions, but only when folding is cooperative. We translate the definition of functional stability into equations and perform simulations of the thermodynamic data, which lend support to this model. The ability to increase the melting temperature of tertiary structure by strengthening base-pairing interactions separate from tertiary interactions provides a simple means for evolving thermostability in functional RNAs.

Inflammatory Blues Turns Velvet Skin Into Rawhide

The directed homing of leukocytes is crucial during immune surveillance or inflammation and is accomplished by a complex cooperation of signaling and adhesion molecules.1,2 Its pathophysiologic relevance is exemplified by various inflammatory diseases such as atherosclerosis.3 At present, the course of leukocyte trafficking is well established through the classical multistep cascade of initial tethering and rolling of leukocytes over vascular endothelium, followed by firm adhesion and their subsequent spreading and transendothelial migration.1,2 Leukocyte rolling is mediated by a subgroup of C-type lectins; E-, L-, and P-selectin, through shear-resistant binding to particular cell-surface carbohydrates. E- and L-selectin are expressed on activated endothelial cells and most leukocytes, respectively, while P-selectin is stored in the secretory compartments of platelets and endothelial cells to become rapidly upregulated on cell activation. The best characterized ligand for selectins is P-selectin glycoprotein ligand-1 (PSGL-1),4 a heavily posttranslationally modified homodimeric transmembrane glycoprotein. PSGL-1 contains functionally essential sialylated and fucosylated carbohydrate moieties, known as sialyl Lewis X (sLex) groups, and is expressed on blood cells such as neutrophils, monocytes, and platelets.5 The importance of PSGL-1 for cell recruitment is highlighted by studies using transgenic mice deficient in PSGL-1, which revealed a crucial contribution to P-selectin–dependent rolling on inflamed endothelium, indicating a function of PSGL-1 in early inflammatory responses.6 Presented by endothelium-bound leukocytes, PSGL-1 supports the initial L-selectin–dependent tethering of blood-borne leukocytes to the already adherent leukocytes at the inflamed vessel wall followed by E-selectin-mediated rolling on the endothelium.7 Besides supporting leukocyte–endothelium interactions, PSGL-1 promotes the P-selectin–dependent initial tethering of platelets to monocytes8,9 followed by more stable interactions through integrins expressed on both platelets and monocytes. In addition, the interaction between PSGL-1 and P-selectin also plays an essential role in the delivery …

The Referendum in Montenegro: The EU?s ?Postmodern Diplomacy?

This article analyses the referendum on independence in Montenegro 21 May 2006. It argues that the 2002 EU-brokered State Union of Serbia and Montenegro by 2006 had become a non-functional state and a source of discord which distracted attention from the urgently needed reform processes in both republics. Due to the deep splits in Montenegro over independence, a new EU engagement to help resolve the issue once and for all and in a stable and democratic way, was needed. The pre-referendum negotiations succeeded due to skilful diplomacy and insistence on European values and principles in the legislative framework. As a result, the referendum proved that even the most contentious and sensitive issues can be resolved with democratic means in the Western Balkans. The EU engagement in this case could be characterized as ?postmodern diplomacy?, because the engagement was aimed directly to an internal situation in another country; because the main asset provided was European legal and political principles; and because the EU included other international organizations (the CoE and OSCE) in the negotiations.

Dominance of the α1B-Adrenergic Receptor and its Subcellular Localization in Human and TRAMP Prostate Cancer Cell Lines

The function and distribution of α1-adrenergic receptor (AR) subtypes in prostate cancer cells is well characterized. Previous studies have used RNA localization or low-avidity antibodies in tissue or cell lines to determine the α1-AR subtype and suggested that the α1 A-AR is dominant. Two androgen-insensitive, human metastatic cancer cell lines DU145 and PC3 were used as well as the mouse TRAMP C1-C3 primary and clonal cell lines. The density of α1-ARs was determined by saturation binding and the distribution of the different α1-AR subtypes was examined by competition-binding experiments. In contrast to previous studies, the major α1-AR subtype in DU145, PC3 and all of the TRAMP cell lines is the α1B-AR. DU145 cells contained 100% of the α1B-AR subtype, whereas PC3 cells were composed of 21% α1 A-AR and 79% α1B-AR. TRAMP cell lines contained between 66% and 79% of the α1B-AR with minor fractions of the other two subtypes. Faster doubling time in the TRAMP cell lines correlated with decreasing α 1B-AR and increasing α1 A- and α1D-AR densities. Transfection with EGFP-tagged α1B-ARs revealed that localization was mainly intracellular, but the majority of the receptors translocated to the cell surface after extended preincubation (18 hr) with either agonist or antagonist. Localization was confirmed by ligand-binding studies and inositol phosphate assays where prolonged preincubation with either agonist and/or antagonist increased the density and function of α 1-ARs, suggesting that the native receptors were mostly intracellular and nonfunctional. Our studies indicate that α1B-ARs are the major α1-AR subtype expressed in DU145, PC3, and all TRAMP cell lines, but most of the receptor is localized in intracellular compartments in a nonfunctional state, which can be rescued upon prolonged incubation with any ligand.

Irrelevant Apoptosis?

Research from two groups suggests that the ability of p53 to stimulate apoptosis in response to DNA damage is independent of its role as a tumor suppressor. p53, which is frequently mutated in human cancers, mediates cellular responses to DNA damage and to oncogenic stress. In wild-type mice, radiation elicits DNA damage-dependent cell death, whereas strains of mice that lack functional p53 are resistant to radiation-induced cell death but rapidly develop radiation-induced lymphomas. Christophorou et al . used transgenic mice that express a 4-hydroxytamoxifen (4-OHT)-sensitive fusion protein that allows p53 to be reversibly switched between functional (in the presence of 4-OHT) and nonfunctional states [homozygous p53ER TAM knockin ( p53 KI/KI ) mice] to investigate the relation between p53’s roles in radiation-dependent apoptosis and as a tumor suppressor. p53 KI/KI mice treated with 4-OHT during the 6 days before irradiation, so that functional p53 was present during irradiation, showed widespread apoptosis of primary lymphoid organs and intestinal epithelium and sustained leukocytopenia (low white blood cell count) but no protection from lymphoma, compared with p53 KI/KI mice that were not treated with 4-OHT. In contrast, p53 KI/KI mice in which p53 function was restored for 6 days starting 8 days after irradiation failed to experience radiation-dependent apoptosis but were protected from radiation-induced lymphomagenesis, with tumor onset delayed about 100 days. The ability of delayed p53 restoration to suppress lymphomagenesis depended on p19 ARF , which is involved in oncogenic signaling to p53, rather than in the DNA damage response. In the second study, Efeyan et al . showed that the protective effect of an extra copy of the p53 gene against spontaneous tumors and tumors following exposure to 3-methyl cholanthrene critically depended on p19 ARF . Thus, both groups conclude that oncogenic signaling to p53, rather than DNA damage-dependent apoptosis, is critical to p53’s role as a tumor suppressor. Berns discusses implications of the research with regard to controlling toxic side effects of cancer therapy. M. A. Christophorou, I. Ringshausen, A. J. Finch, L. Brown Swigart, G. I. Evan, The pathological response to DNA damage does not contribute to p53-mediated tumour suppression. Nature 443 , 214-217 (2006). [PubMed] A. Efeyan, I. Garcia-Cao, D. Herranz, S. Velasco-Miguel, M. Serrano, Policing of oncogene activity by p53. Nature 443 , 159 (2006). [PubMed] A. Berns, Can less be more for p53? Nature 443 , 153-154 (2006). [PubMed]

The maxi K+ channel of human myometrium reveals a split personality

The maxi K⁺ channel of human myometrium reveals a split personality

Comparative Study on the Changes in Photosynthetic Activity of the Homoiochlorophyllous Desiccation-Tolerant Haberlea Rhodopensis and Desiccation-Sensitive Spinach Leaves During Desiccation and Rehydration

The functional peculiarities and responses of the photosynthetic system in the flowering homoiochlorophyllous desiccation-tolerant (HDT) Haberlea rhodopensis and the non-desiccation-tolerant spinach were compared during desiccation and rehydration. Increasing rate of water loss clearly modifies the kinetic parameters of fluorescence induction, thermoluminescence emission, far-red induced P700 oxidation and oxygen evolution in the leaves of both species. The values of these parameters returned nearly to the control level after 24 h rehydration only of the leaves of HDT plant. PS II was converted in a non-functional state in desiccated spinach in accordance with the changes in membrane permeability, malondialdehyde, proline and H(2)O(2) contents. Moreover, our data showed a strong reduction of the total number of PS II centers in Haberlea without any changes in the energetics of the charge recombination. We consider this observation, together with the previously reported unusually high temperature of B-band (S(2)Q(B)-) emission of Haberlea to reflect some specific adaptive characteristics of the photosynthetic system. As far as we know this is the first time when such adaptive characteristics and mechanism of the photosynthetic system of a flowering HDT higher plant is described. These features of Haberlea can explain the fast recovery of its photosynthesis after desiccation, which enable this HDT plant to rapidly take advantage of frequent changes in water availability.

Kinetic Partitioning During Folding of the p53 DNA Binding Domain

The DNA-binding domain (DBD) of wild-type p53 loses DNA binding activity spontaneously at 37 degrees C in vitro, despite being thermodynamically stable at this temperature. We test the hypothesis that this property is due to kinetic misfolding of DBD. Interrupted folding experiments and chevron analysis show that native molecules are formed via four tracks (a-d) under strongly native conditions. Folding half-lives of tracks a-d are 7.8 seconds, 50 seconds, 5.3 minutes and more than five hours, respectively, in 0.3M urea (10 degrees C). Approximately equal fractions of molecules fold through each track in zero denaturant, but above 2.0M urea approximately 90% fold via track c. A kinetic mechanism consisting of two parallel folding channels (fast and slow) is proposed. Each channel populates an on-pathway intermediate that can misfold to form an aggregation-prone, dead-end species. Track a represents direct folding through the fast channel. Track b proceeds through the fast channel but via the off-pathway state. Track c corresponds to folding via the slow channel, primarily through the off-pathway state. Track d proceeds by way of an even slower, uncharacterized route. We postulate that activity loss is caused by partitioning to the slower tracks, and that structural unfolding limits this process. In support of this view, tumorigenic hot-spot mutants G245S, R249S and R282Q accelerate unfolding rates but have no affect on folding kinetics. We suggest that these and other destabilizing mutants facilitate loss of p53 function by causing DBD to cycle unusually rapidly between folded and unfolded states. A significant fraction of DBD molecules become effectively trapped in a non-functional state with each unfolding-folding cycle.

Low pH-induced conformational changes in 33 kD protein of photosystem II

33 kD protein, located on the lumen side of thylakoid membranes, is one of three extrinsic proteins of photosystem II (PS II). Previous study showed that NBS modification of W241, the only tryptophan in 33 kD protein, is helpful for understanding the function of W241 in maintaining functional conformation of 33 kD protein. In this paper, studies of both circular dichroism and fluorescence spectra showed that upon decreasing pH from 6.2 to 2.5, the conformation of soluble 33 kD protein changed significantly, with an increase or a decrease in percentage of random coil or α-helix and turns. The changes in secondary structures of this protein are pH reversible. After NBS modification at pH 2.5, the conformational change of 33 kD protein was kept fixed. The CD ellipticity at 200 nm for NBS-modified 33 kD protein is much lower than that for control, indicating that the unfolding degree of 33 kD protein was enhanced after the NBS modification. Moreover, the conformational flexibility is lost in NBS-modified 33 kD protein, and the conformational change becomes pH irreversible, indicating that NBS modification blocked the reversibility of conformational change of 33 kD protein. The specific binding capability of NBS-modified 33 kD protein is much lower than that of low pH-treated control. Furthermore, the rebinding of modified protein on PS II membranes cannot restore the activity of oxygen evolution. We suggest that it is low pH but not NBS modification of W241 that leads to the conformational change of 33 kD protein from one functional to another non-functional state. The significant capability of proton transport of 33 kD protein is discussed.

A t-SNARE of the endocytic pathway must be activated for fusion.

The t-SNARE in a late Golgi compartment (Tlg2p) syntaxin is required for endocytosis and localization of cycling proteins to the late Golgi compartment in yeast. We show here that Tlg2p assembles with two light chains, Tlg1p and Vti1p, to form a functional t-SNARE that mediates fusion, specifically with the v-SNAREs Snc1p and Snc2p. In vitro, this t-SNARE is inert, locked in a nonfunctional state, unless it is activated for fusion. Activation can be mediated by a peptide derived from the v-SNARE, which likely bypasses additional regulatory proteins in the cell. Locking t-SNAREs creates the potential for spatial and temporal regulation of fusion by signaling processes that unleash their fusion capacity.

Ceramide generation in situ alters leukocyte cytoskeletal organization and beta 2-integrin function and causes complete degranulation.

Ceramide levels increase in activated polymorphonuclear neutrophils, and here we show that endogenous ceramide induced degranulation and superoxide generation and increased surface beta(2)-integrin expression. Ceramide accumulation reveals a bifurcation in integrin function, as it abolished agonist-induced adhesion to planar surfaces, yet had little effect on homotypic aggregation. We increased cellular ceramide content by treating polymorphonuclear neutrophils with sphingomyelinase C and controlled for loss of sphingomyelin by pretreatment with sphingomyelinase D to generate ceramide phosphate, which is not a substrate for sphingomyelinase C. Pretreatment with the latter enzyme blocked all the effects of sphingomyelinase C. Ceramide generation caused a Ca(2+) flux and complete degranulation of both primary and secondary granules and increased surface beta(2)-integrin expression. These integrins were in a nonfunctional state, and subsequent activation with platelet-activating factor or formyl-methionyl-leucyl-phenylalanine induced beta(2)-integrin-dependent homotypic aggregation. However, these cells were completely unable to adhere to surfaces via beta(2)-integrins. This was not due to a defect in the integrins themselves because the active conformation could be achieved by cation switching. Rather, ceramide affected cytoskeletal organization and inside-out signaling, leading to affinity maturation. Cytochalasin D induced the same disparity between aggregation and surface adhesion. We conclude that ceramide affects F-actin rearrangement, leading to massive degranulation, and reveals differences in beta(2)-integrin-mediated adhesive events.

Executive summary: stages of reproductive aging workshop (STRAW)

A select group of investigators attended a structured workshop, the Stages of Reproductive Aging Workshop (STRAW), at Park City, Utah, USA, in July 2001, which addressed the need in women for a staging system as well as the confusing nomenclature for the reproductive years.

Disc mobility and arthroscopic condition of the temporomandibular joint associated with long-term mandibular discontinuity

Purpose: The present study aimed to explore disc mobility and arthroscopically diagnosed morphologic changes in the temporomandibular joint (TMJ) associated with long-term mandibular discontinuity. Patients and Methods: Twelve patients (24 TMJs) who had undergone mandibulectomy including the unilateral condyle or segmental mandibulectomy without mandibular reconstruction, more than 8 months before this study were examined. The 24 TMJs were classified into 3 groups as follows: group 1, TMJs with a major mandibular fragment including the mandibular body (n = 11); group 2, TMJs with a mandibular ramus including the coronoid process (n = 5); and group 3, TMJs with only a condylar process, a mandibular ramus not including the coronoid process, or without a condyle (n = 8). Disc mobility was evaluated by magnetic resonance imaging, and arthroscopic observation of the superior joint compartment (SJC) was performed in all TMJs. The relationship between disc mobility and the arthroscopic findings was also studied. Results: The frequency of immobile discs differed significantly among groups 1 (0%), 2 (40.0%), and 3 (100%). Arthroscopic findings were normal in all SJCs of group 1. Various types of fibrous adhesions were observed in 40.0% of group 2 and in 75.0% of group 3. The development of fibrous adhesions in the SJC was significantly related to the presence of an immobile disc. Conclusions: Long-term immobilization of the TMJ in a nonfunctional state seems to promote the development of fibrous adhesions in the SJC. Preservation of the mandibular fragment including the coronoid process, during mandibulectomy appears to contribute to postoperative TMJ mobility. To ensure recovery of a physiologic TMJ after mandibulectomy, it seems important to re-establish TMJ mobility by establishing mandibular continuity as soon as possible. © 2001 American Association of Oral and Maxillofacial Surgeons

Spontaneous inactivation of human tryptase involves conformational changes consistent with conversion of the active site to a zymogen-like structure.

The conformational changes accompanying spontaneous inactivation and dextran sulfate (DS) mediated reactivation of the serine protease human tryptase were investigated by analysis of (i) intrinsic fluorescence, (ii) inhibitor binding, and (iii) catalytic efficiency. Spontaneous inactivation produced a marked decrease in fluorescence emission intensity that was reversed by the addition of DS. Fluorescence decreases at high (4.0 microM) and low (0.1 microM) tryptase concentrations were similar at early times and coincided with loss of enzymatic activity but deviated significantly from activity loss at later times by showing a difference in the extent of change. The fluorescence losses were best described by a two-step kinetic model in which the major decrease correlated to activity loss (t1/2 of 4.3 min in 0.2 M NaCl, pH 6.8, 30 degrees C) and was followed by a further decrease (t1/2 approximately 60 min) whose extent differed with tryptase concentration. The ability to bind the competitive inhibitor p-aminobenzamidine was reversibly lost upon spontaneous inactivation, providing evidence for conformational changes affecting the major substrate binding site (S1-pocket). Estimation of catalytic efficiency using an active site titrant showed that the specific activity of tryptase remained unchanged upon inactivation and reactivation. Return of enzymatic activity, intrinsic fluorescence, and the S1 pocket appeared to occur in the same time frame (t1/2 approximately 3 min). These studies indicate that spontaneous inactivation involves reversible changes which convert the active site to a nonfunctional state. The association of activity loss with an intrinsic fluorescence decrease and loss of the S1-pocket is consistent with the disruption of a critical ionic bond at the active site. Formation of this ionic bond is the basis of zymogen activation for the chymotrypsin family of serine proteases.

β-Catenin is a Target for Extracellular Signals Controlling Cadherin Function: The Neurocan-GalNAcPTase Connection

Publisher Summary This chapter describes how the association of cadherin with the actin-containing cytoskeleton is rapidly uncoupled, by a signal pathway triggered, by the interaction between a cell surface glycosyltransferase and its proteoglycan ligand, neurocan. The adhesion molecules that participate in these interactions belong to a limited number of families: the cadherins, N-CAM family members, and integrins. Cadherin-mediated adhesion is one of the key processes guiding the cell-cell interactions essential to the formation and maintenance of regionally specific tissue organization. The adhesive function of N- and E-cadherin is dependent on their association with the cytoskeleton. β-Catenin has the potential to play an important role in this process, as its tyrosine phosphorylation/dephosphorylation can rapidly switch cadherin between functional and nonfunctional states. The neurocan/GalNAcPTase pair is one of the several ligand/receptor interactions that can trigger this process. The fine-tuning of many morphogenetic processes may depend on the temporal and spatial distribution of these ligand/receptor pairs. This chapter examines how other receptor-ligand interactions affect the phosphorylation state of β-catenin, and finally it integrates the role of β-catenin in modulating cadherin function, with the key roles, that it plays in the wingless signaling pathway, its recently described transcriptional activation activity, and its association with the APC tumor suppressor protein, important pathways that regulate development, and neoplasia.

Evolving stroke and the ischemic penumbra.

Deterioration of the neurologic deficits that occur hours or even days after the initial presentation is a well-recognized feature of ischemic stroke. In several recent series of prospectively evaluated ischemic stroke patients, the frequency of neurologic worsening ranged from 26 to 43%, and the rate of deterioration was similar in patients evaluated either within 6 to 8 hours or within 1 to 2 days after the onset of symptoms. [1-3] These rates of progression were similar to those previously observed by Jones and Millikan [4] and Jones et al. [5] in patients with either carotid or vertebrobasilar arterial occlusions who were evaluated within 36 hours of stroke onset, although a higher percentage of vertebrobasilar system stroke patients deteriorated at a later time. The definition of neurologic worsening after stroke has varied from study to study, making direct comparisons difficult. With the advent of valid, reproducible neurologic scoring scales, such as the NIH Stroke Scale and the Scandinavian Stroke Scale, a more precise perspective on short-term clinical worsening after acute ischemic stroke will be available from the data that the current plethora of acute stroke intervention trials will provide. [6] The factors that make the deficit worsen are not known. The object of this review is to advance the concept that, in some patients with ischemic stroke, the progression of the neurologic deficits shortly after the ictus relates to the deterioration of the local circulation in which an initially ischemic, but functional, region progresses to a non-functional state and ultimately to infarction. The concept of evolving or progressing stroke initially appeared in the 1950s. [7,8] Several mechanisms potentially contribute to the phenomenon of evolving stroke, and early authors emphasized thrombus propagation despite scarce confirmation of this hypothesis. [9] Other potential mechanisms for the increasing clinical deterioration that may occur during the …

Involvement of silent synapses in the induction of long-term potentiation and long-term depression in neocortical and hippocampal neurons

Changes in the latency of small excitatory postsynaptic potentials were observed in association with induction of long-term modifications of synaptic transmission in slices of rat neocortex and guinea-pig hippocampus. After potentiation response latency decreased in 3/10 cases in the neocortex and in 6/24 cases in the hippocampus, and increased after depression in 4/8 cases in the neocortex. These latency changes could not be attributed to changes in presynaptic fibre excitability, monosynaptic inhibition, release kinetics or activation kinetics of postsynaptic ion channels. We conclude therefore that potentiation led to the activation of previously silent synapses of fast-conducting afferents and depression to the inactivation of previously functional synapses. Thus, neocortical and hippocampal synapses can be in a non-functional state, and regimes that induce long-term potentiation and depression not only change the efficacy of synapses but also alter their functional state.

Conformational trapping in a membrane environment: a regulatory mechanism for protein activity?

Functional regulation of proteins is central to living organisms. Here it is shown that a nonfunctional conformational state of a polypeptide can be kinetically trapped in a lipid bilayer environment. This state is a metastable structure that is stable for weeks just above the phase transition temperature of the lipid. When the samples are incubated for several days at 68 degrees C, 50% of the trapped conformation converts to the minimum-energy functional state. This result suggests the possibility that another mechanism for functional regulation of protein activity may be available for membrane proteins: that cells may insert proteins into membranes in inactive states pending the biological demand for protein function.

Thermoluminescence studies on the function of Photosystem II in the desiccation tolerant lichen Cladonia convoluta

The effect of desiccation and rehydration on the function of Photosystem II has been studied in the desiccation tolerant lichen Cladonia convoluta by thermoluminescence. We have shown that in functional fully hydrated thalli thermoluminescence signals can be observed from the recombination of the S2(3)QB (-) (B band), S2QA (-) (Q band), Tyr-D(+)QA (-) (C band) and Tyr-Z(+)(His(+))QA (-) (A band) charge stabilization states. These thermoluminescence signals are completely absent in desiccated thalli, but rapidly reappear on rehydration. Flash-induced oscillation in the amplitude of the thermoluminescence band from the S2(3)QB (-) recombination shows the usual pattern with maxima after 2 and 6 flashes when rehydration takes place in light. However, after rehydration in complete darkness, there is no thermoluminescence emission after the 1 st flash, and the maxima of the subsequent oscillation are shifted to the 3rd and 7th flashes. It is concluded that desiccation of Cladonia convoluta converts PS II into a nonfunctional state. This state is characterized by the lack of stable charge separation and recombination, as well as by a one-electron reduction of the water-oxidizing complex. Restoration of PS II function during rehydration can proceed both in the light and in darkness. After rehydration in the dark, the first charge separation act is utilized in restoring the usual oxidation state of the water-oxidizing comples.