Application of the Marcus theory for analysis of the temperature dependence of the reactions leading to photosynthetic water oxidation: results and implications

The temperature dependence of donor side reactions was analysed within the framework of the Marcus theory of nonadiabatic electron transfer. The following results were obtained for PS II membrane fragments from spinach: (1) the reorganisation energy of P680^+? reduction by Y_Z is of the order of 0.5?eV in samples with a functionally fully competent water oxidising complex (WOC); (2) destruction of the WOC by Tris-washing gives rise to a drastic increase of λ to values of the order of 1.6?eV; (3) the reorganisation energies of the oxidation steps in the WOC are dependent, on the redox states S _i with values of about 0.6?eV for the reactions Y_Z ^OX S ₀→Y_Z S ₁ and Y_Z ^OX S ₁→Y_Z S ₂, 1.6?eV for the reaction Y_Z ^OX S ₂→Y_Z S ₃ and 1.1?eV (above a characteristic temperature u_c of about 6??°C) for the reaction Y_Z ^OX S ₃→→Y_Z S ₀+O₂. Using an empirical rate constant-distance relationship, the van der Waals distance between Y_Z and P680 was found to be about 10?Å, independent of the presence or absence of the WOC, whereas the distance between Y_Z and the manganese cluster in the WOC was ≥15?Å. Based on the calculated activation energies the environment of Y_Z is inferred to be almost "dry" and hydrophobic when the WOC is intact but becomes enriched with water molecules after WOC destruction. Furthermore, it is concluded that the transition S ₂→S ₃ is an electron transfer reaction gated by a conformational change, i.e. it comprises significant structural changes of functional relevance. Measurements of kinetic H/D isotope exchange effects support the idea that none of these reactions is gated by the break of a covalent O-H bond. The implications of these findings for the mechanism of water oxidation are discussed.     

A cyclobutane thymine–N4-methylcytosine dimer is resistant to hydrolysis but strongly blocks DNA synthesis

Exposure of DNA to ultraviolet light produces harmful crosslinks between adjacent pyrimidine bases, to form cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6–4)pyrimidone photoproducts. The CPD is frequently formed, and its repair mechanisms have been exclusively studied by using a CPD formed at a TT site. On the other hand, biochemical analyses using CPDs formed within cytosine-containing sequence contexts are practically difficult, because saturated cytosine easily undergoes hydrolytic deamination. Here, we found that N-alkylation of the exocyclic amino group of 2′-deoxycytidine prevents hydrolysis in CPD formation, and an N-methylated cytosine-containing CPD was stable enough to be derivatized into its phosphoramidite building block and incorporated into oligonucleotides. Kinetic studies of the CPD-containing oligonucleotide indicated that its lifetime under physiological conditions is relatively long (∼7 days). In biochemical analyses using human DNA polymerase η, incorporation of TMP opposite the N-methylcytosine moiety of the CPD was clearly detected, in addition to dGMP incorporation, and the incorrect TMP incorporation blocked DNA synthesis. The thermodynamic parameters confirmed the formation of this unusual base pair.     

Impact of initial conditions on extant microbial kinetic parameter estimates: application to chlorinated ethene dehalorespiration

Monod kinetics are the foundation of mathematical models of many environmentally important biological processes, including the dehalorespiration of chlorinated ethene groundwater contaminants. The Monod parameters—q _max, the maximum specific substrate utilization rate, and K _S, the half-saturation constant—are typically estimated in batch assays, which are superficially simple to prepare and maintain. However, if initial conditions in batch assays are not chosen carefully, it is unlikely that the estimated parameter values will be meaningful because they do not reflect microbial activity in the environmental system of interest, and/or they are not mathematically identifiable. The estimation of q _max and K _S values that are highly correlated undoubtedly contributes significantly to the wide range in reported parameter values and may undermine efforts to use mathematical models to demonstrate the occurrence of natural attenuation or predict the performance of engineered bioremediation approaches. In this study, a series of experimental and theoretical batch kinetic assays were conducted using the tetrachloroethene-respirer Desulfuromonas michiganensis to systematically evaluate the effects of initial batch assay conditions, expressed as the initial substrate (S ₀)-to-initial biomass concentration (X ₀) ratio (S ₀/X ₀) and the S ₀/K _S ratio on parameter correlation. An iterative approach to obtain meaningful Monod parameter estimates was developed and validated using three different strains and can be broadly applied to a range of other substrates and populations. While the S ₀/X ₀ ratio is critical to obtaining kinetic parameter estimates that reflect in situ microbial activity, this study shows that optimization of the S ₀/K _S ratio is key to minimizing Monod parameter correlation.     

Simultaneous estimation ofV max,Km, and the rate of endogenous substrate production (R) from substrate depletion data

The nonlinear and 3 linearized forms of the integrated Michaelis-Menten equation were evaluated for their ability to provide reliable estimates of uptake kinetic parameters, when the initial substrate concentration (S₀) is not error-free. Of the 3 linearized forms, the one where t/(S₀–S) is regressed against ln(S₀/S)/(S₀–S) gave estimates ofV _max and K_m closest to the true population means of these parameters. Further, this linearization was the least sensitive of the 3 to errors (±1%) in S₀. Our results illustrate the danger of relying on r² values for choosing among the 3 linearized forms of the integrated Michaelis-Menten equation. Nonlinear regression analysis of progress curve data, when S₀ is not free of error, was superior to even the best of the 3 linearized forms. The integrated Michaelis-Menten equation should not be used to estimateV _max and K_m when substrate production occurs concomitant with consumption of added substrate. We propose the use of a new equation for estimation of these parameters along with a parameter describing endogenous substrate production (R) for kinetic studies done with samples from natural habitats, in which the substrate of interest is an intermediate. The application of this new equation was illustrated for both simulated data and previously obtained H₂ depletion data. The only means by whichV _max, K_m, and R may be evaluated from progress curve data using this new equation is via nonlinear regression, since a linearized form of this equation could not be derived. Mathematical components of computer programs written for fitting data to either of the above nonlinear models using nonlinear least squares analysis are presented.     

Interaction between tyrosineZ and substrate water in active photosystem II

In the field of photosynthetic water oxidation it has been under debate whether Tyrosine_Z (Tyr_Z) acts as a hydrogen or an electron acceptor from water. In the former concept, direct contact of Tyr_Z with substrate water has been assumed. However, there is no direct evidence for the interaction between Tyr_Z and substrate water in active Photosystem II (PSII), instead most experiments have been performed on inhibited PSII. Here, this problem is tackled in active PSII by combining low temperature EPR measurements and quantum chemistry calculations. EPR measurements observed that the maximum yield of Tyr_Z oxidation at cryogenic temperature in the S₀ and S₁ states was around neutral pH and was essentially pH-independent. The yield of Tyr_Z oxidation decreased at acidic and alkaline pH, with pKs at 4.7-4.9 and 7.7, respectively. The observed pH-dependent parts at low and high values of pH can be explained as due to sample inactivation, rather than active PSII. The reduction kinetics of Tyr_Z^· in the S₀ and S₁ states were pH independent at pH range from 4.5 to 8. Therefore, the change of the pH in bulk solution probably has no effect on the Tyr_Z oxidation and Tyr_Z^· reduction at cryogenic temperature in the S₀ and S₁ states of the active PSII. Theoretical calculations indicate that Tyr_Z becomes more difficult to oxidize when a H₂O molecule interacts directly with it. It is suggested that Tyr_Z is probably located in a hydrophobic environment with no direct interaction with the substrate H₂O in active PSII. These results provide new insights on the function and mechanism of water oxidation in PSII.     

S-adenosylhomocysteine hydrolase from hamster liver: Purification and kinetic properties

3-Deazaadenosine is both an inhibitor of and a substrate for S-adenosylhomocysteine hydrolase. Its administration to rats results in the accumulation of both S-adenosylhomocysteine and 3-deazaadenosylhomocysteine in the liver and other tissues. In hamsters, however, the administration of 3-deazaadenosine results only in the accumulation of 3-deazaadenosylhomocysteine (P. K. Chiang and G. L. Cantoni (1979) Biochem. Pharmacol. 28, 1897). In order to investigate the possible reasons for this difference, S-adenosylhomocysteine hydrolase from hamster liver has been purified to homogeneity and some of its kinetic and physical parameters have been determined. The molecular weight of the native enzyme is 200,000 with a subunit molecular weight of 48,000. The K_m's for adenosine and 3-deazaadenosine are about 1.0 μm, and the V_max's are also similar. The K_m for S-adenosylhomocysteine is 1.0 μm, or more than 10 times smaller than the K_m of the rat liver enzyme. This difference in K_m value may explain the differences in the response of rat and hamster liver to the administration of 3-deazaadenosine. S-Adenosylhomocysteine hydrolase from hamster liver exhibits an interesting kinetic property in that its activity can be affected bimodally by either adenosine or adenosine Anal.ogs. At very low concentrations of these analogs, the activity of S-adenosylhomocysteine hydrolase can be stimulated by 10–30%, and at higher concentrations these same analogs become competitive inhibitors.     

Effect of sequence and metal ions on UVB-induced anti cyclobutane pyrimidine dimer formation in human telomeric DNA sequences

Irradiation of G-quadruplex forming human telomeric DNA with ultraviolet B (UVB) light results in the formation of anti cyclobutane pyrimidine dimers (CPDs) between loop 1 and loop 3 in the presence of potassium ions but not sodium ions. This was unexpected because the sequences involved favor the nonphotoreactive hybrid conformations in K⁺ solution, whereas a potentially photoreactive basket conformation is favored in Na⁺ solution. To account for these contradictory results, it was proposed that the loops are too far apart in the basket conformation in Na⁺ solution but close enough in a two G-tetrad basket-like form 3 conformation that can form in K⁺ solution. In the current study, Na⁺ was still found to inhibit anti CPD formation in sequences designed to stabilize the form 3 conformation. Furthermore, anti CPD formation in K⁺ solution was slower for the sequence previously shown to exist primarily in the proposed photoreactive form 3 conformation than the sequence shown to exist primarily in a nonphotoreactive hybrid conformation. These results suggest that the form 3 conformation is not the principal photoreactive conformation, and that G-quadruplexes in K⁺ solution are dynamic and able to access photoreactive conformations more easily than in Na⁺ solution.     

NMR structure of the DNA decamer duplex containing double T*G mismatches of cis-syn cyclobutane pyrimidine dimer: implications for DNA damage recognition by the XPC-hHR23B complex

The cis–syn cyclobutane pyrimidine dimer (CPD) is a cytotoxic, mutagenic and carcinogenic DNA photoproduct and is repaired by the nucleotide excision repair (NER) pathway in mammalian cells. The XPC–hHR23B complex as the initiator of global genomic NER binds to sites of certain kinds of DNA damage. Although CPDs are rarely recognized by the XPC–hHR23B complex, the presence of mismatched bases opposite a CPD significantly increased the binding affinity of the XPC–hHR23B complex to the CPD. In order to decipher the properties of the DNA structures that determine the binding affinity for XPC–hHR23B to DNA, we carried out structural analyses of the various types of CPDs by NMR spectroscopy. The DNA duplex which contains a single 3′ T·G wobble pair in a CPD (CPD/GA duplex) induces little conformational distortion. However, severe distortion of the helical conformation occurs when a CPD contains double T·G wobble pairs (CPD/GG duplex) even though the T residues of the CPD form stable hydrogen bonds with the opposite G residues. The helical bending angle of the CPD/GG duplex was larger than those of the CPD/GA duplex and properly matched CPD/AA duplex. The fluctuation of the backbone conformation and significant changes in the widths of the major and minor grooves at the double T·G wobble paired site were also observed in the CPD/GG duplex. These structural features were also found in a duplex that contains the (6–4) adduct, which is efficiently recognized by the XPC–hHR23B complex. Thus, we suggest that the unique structural features of the DNA double helix (that is, helical bending, flexible backbone conformation, and significant changes of the major and/or minor grooves) might be important factors in determining the binding affinity of the XPC–hHR23B complex to DNA.     

Rapid Deamination of Cyclobutane Pyrimidine Dimer Photoproducts at TCG Sites in a Translationally and Rotationally Positioned Nucleosome in Vivo

Sunlight-induced C to T mutation hot spots in skin cancers occur primarily at methylated CpG sites that coincide with sites of UV-induced cyclobutane pyrimidine dimer (CPD) formation. The C and 5-methyl-C in CPDs are not stable and deaminate to U and T, respectively, which leads to the insertion of A by the DNA damage bypass polymerase η, thereby defining a probable mechanism for the origin of UV-induced C to T mutations. Deamination rates for T^mCG CPDs have been found to vary 12-fold with rotational position in a nucleosome in vitro. To determine the influence of nucleosome structure on deamination rates in vivo, we determined the deamination rates of CPDs at TCG sites in a stably positioned nucleosome within the FOS promoter in HeLa cells. A procedure for in vivo hydroxyl radical footprinting with Fe-EDTA was developed, and, together with results from a cytosine methylation protection assay, we determined the translational and rotational positions of the TCG sites. Consistent with the in vitro observations, deamination was slower for one CPD located at an intermediate rotational position compared with two other sites located at outside positions, and all were much faster than for CPDs at non-TCG sites. Photoproduct formation was also highly suppressed at one site, possibly due to its interaction with a histone tail. Thus, it was shown that CPDs of TCG sites deaminate the fastest in vivo and that nucleosomes can modulate both their formation and deamination, which could contribute to the UV mutation hot spots and cold spots.     

Acceleration of 5-Methylcytosine Deamination in Cyclobutane Dimers by G and Its Implications for UV-Induced C-to-T Mutation Hotspots

Sunlight-induced C→T mutation hotspots occur most frequently at methylated CpG sites in tumor suppressor genes and are thought to arise from translesion synthesis past deaminated cyclobutane pyrimidine dimers (CPDs). While it is known that methylation enhances CPD formation in sunlight, little is known about the effect of methylation and sequence context on the deamination of 5-methylcytosine (^mC) and its contribution to mutagenesis at these hotspots. Using an enzymatic method, we have determined the yields and deamination rates of C and ^mC in CPDs and find that the frequency of UVB-induced CPDs correlates with the oxidation potential of the flanking bases. We also found that the deamination of T^mC and ^mCT CPDs is about 25-fold faster when flanked by G's than by A's, C's or T's in duplex DNA and appears to involve catalysis by the O6 group of guanine. In contrast, the first deamination of either C or ^mC in AC^mCG with a flanking G was much slower (t_1/2 > 250 h) and rate limiting, while the second deamination was much faster. The observation that C^mCG dimers deaminate very slowly but at the same time correlate with C→T mutation hotspots suggests that their repair must be slow enough to allow sufficient time for deamination. There are, however, a greater number of single C→T mutations than CC→TT mutations at C^mCG sites even though the second deamination is very fast, which could reflect faster repair of doubly deaminated dimers.     

Test of Normality for Integrated Change Point Detection and Mixture Modeling

Single-molecule data often show step-like changes in the quantity measured between constant levels. Analysis of this data consists of detecting the steps, i.e., change point detection (CPD), and determining the levels, i.e., clustering. We describe a novel algorithm which integrates these two analyses, based on a statistical test of a normal distribution. The test of normality (TON) algorithm integrates statistical CPD with gaussian mixture model clustering. We used TON with both simulated data and ion channel patch-clamp recordings. It performed well with simulated data except at a high signal-to-noise ratio and when the frequency of steps was high compared to the sampling frequency. TON has advantages over separate CPD and mixture modeling algorithms, especially for complex single-molecule data. This was illustrated by its application to the maxichannel, an ion channel with multiple subconductance states.     

Research of the (E/Z)-isomerization of carotenoids in Pécs since the 1970s

Geometrical configuration of the polyene chain of approximately 40 mono- and di-cis carotenoids was determined from 1970 through 1990. Subsequently, the kinetic, equilibrium and thermodynamic parameters (k, K, A, E_A, ΔH^#, ΔG^#, ΔS^#) of the reversible thermal isomerization of several symmetrical and unsymmetrical carotenoids were calculated. The rate of the iodine-catalyzed photoisomerization of (all-E)-, (9Z)- and (13Z)-zeaxanthin was compared and the ‘specific rate’ (per unit light energy at given wavelengths) of the iodine-catalyzed photoisomerization for several (13Z)-carotenoids was investigated. As the missing links of the biosynthetic pathway of paprika-carotenoids, carotenoids containing new end groups were isolated; their sterically unhindered mono-cis isomers were also prepared and their geometrical configuration was determined. The investigation concentrated on the substrate specificity of the enzyme violaxanthin-deepoxidase, the light-induced formation of (13Z)-violaxanthin in green leaves, the binding of xanthophylls to the bulk light-harvesting complex (LHC) of photosystem II in higher plants, the biochemical basis of color as an aesthetic quality in Citrus-fruits and the (9Z)-epoxycarotenoid cleavage enzyme activity for ABA biosynthesis. Recently (9Z)-capsanthin-5,6-epoxide and capsoneoxanthin, two novel carotenoids have been isolated from natural sources.     

Stereochemical control of yeast reductions. 2. Quantitative treatment of the kinetics of competing enzyme systems for a single substrate

Quantitative expressions have been developed for systems such as yeast reductions where competing enzymes act on one substrate to yield two enantiomeric products. These expressions relate the observed stereochemical variables, the extent of conversion (C), the optical purity expressed as enantiomeric excess (ee), and the initial substrate concentration (A₀) to the kinetic parameters K_R and K_S (apparent Michaelis constants) and y ($\text{V}{}_{\mathrm{R}}\text{V}{}_{\mathrm{S}}$, the ratio of maximal velocities) of such competing enzymes. The expressions have been experimentally verified using a purified competing enzyme system of l- and d-lactic dehydrogenases. Furthermore, the enantioselective reduction of β-keto esters by intact yeast cells has been examined by means of this kinetic analysis.     

Yield and kinetic constants estimation in the production of hydroxy fatty acids from oleic acid in a bioreactor by Pseudomonas aeruginosa 42A2

We modelled the production of hydroxy fatty acids from oleic acid by Pseudomonas aeruginosa 42A2 in a bioreactor with a non-dispersive aeration system. First, we designed an adapted wetted-wall gas-absorption column, offering a k _La value of 39.9 h^?1, to enhance oxygen absorption in the culture media and prevent foam formation. Then, we analysed different kinetic models to simulate the yield coefficients and the kinetic constants in this bacterial transformation. Monod model fitting (μ _max1?=?0.51 h^?1, K _S1?=?1.60 C-mol l^?1, μ _max2?=?0.12 h^?1, K _S2?=?0.035 C-mol l^?1, and k ₂?=?0.033 h^?1) showed a good accuracy with the experimental data sets and was chosen for its simplicity. Lastly, mass balances were carried out to establish the stoichiometry of this biotransformation with the following yield coefficients, Υ _X/OA, Υ _{X/(10S)-HPOME} and Υ _{(10S)-HPOME/(7S10S)-HPOME} of 0.172, 0.347 and 2.388 C-mol C-mol^?1, respectively.     

The relative quantities and catalytic activities of enzymes produced by alleles at the alcohol dehydrogenase locus in Drosophila melanogaster

We have examined the kinetic properties of enzymes produced by the electrophoretically fast (F) and slow (S) alleles at the alcohol dehydrogenase locus in a polymorphic laboratory population of Drosophila melanogaster. The product of the F allele has approximately twice the specific activity of the product of the S allele. We have estimated four Michaelis constants (K _ethanol, K _NAD, K′_ethanol, and K′_NAD) and have found no significant difference between the major (ADH-5) isozyme produced by homozygotes for the two alleles. The relative amounts of enzyme produced by the homozygotes were estimated by the method of Laurell (1966), and again no significant differences were found. It appears that the difference in specific activities can be explained solely in terms of relative catalytic efficiency. In a series of laboratory stocks, those fixed for the F allele tend to produce more enzyme than those fixed for the S allele. These observations do not support the view that the alleles are selectively equivalent.     

Reaction pattern and mechanism of light induced oxidative water splitting in photosynthesis

This mini review is an attempt to briefly summarize our current knowledge on light driven oxidative water splitting in photosynthesis. The reaction leading to molecular oxygen and four protons via photosynthesis comprises thermodynamic and kinetic constraints that require a balanced fine tuning of the reaction coordinates. The mode of coupling between electron (ET) and proton transfer (PT) reactions is shown to be of key mechanistic relevance for the redox turnover of Y_Z and the reactions within the WOC. The WOC is characterized by peculiar energetics of its oxidation steps in the WOC. In all oxygen evolving photosynthetic organisms the redox state S₁ is thermodynamically most stable and therefore this general feature is assumed to be of physiological relevance. Available information on the Gibbs energy differences between the individual redox states S_i+1 and S_i and on the activation energies of their oxidative transitions are used to construct a general reaction coordinate of oxidative water splitting in photosystem II (PS II). Finally, an attempt is presented to cast our current state of knowledge into a mechanism of oxidative water splitting with special emphasis on the formation of the essential O-O bond and the active role of the protein environment in tuning the local proton activity that depends on time and redox state S_i. The O-O linkage is assumed to take place within a multistate equilibrium at the redox level of S₃, comprising both redox isomerism and proton tautomerism. It is proposed that one state, S₃(P), attains an electronic configuration and nuclear geometry that corresponds with a hydrogen bonded peroxide which acts as the entatic state for the generation of complexed molecular oxygen through S₃(P) oxidation by Y_Z^ox.     

A kinetic characterization of the rabbit plasmin isozymes.

Steady-state and presteady-state kinetic parameters for plasmins derived from the two rabbit plasminogen isozymes have been determined. Steady-state kinetic experiments with N-α-tosyl-l-arginine methyl ester indicate that each isozyme has a similar V. Plasmin isozyme 2 has a higher K_m value for this substrate as well as a higher K_i, for the competitive inhibitor, benzamidine-HCl. Presteady-state kinetic experiments, using the p-nitrophenyl esters of p-(methylethylsulfoniummethyl)benzoate, p-(pyridiniummethyl) benzoate, p-(thiouroniummethyl)benzoate and p-(guanidinium)benzoate, indicate that each plasmin has similar rate constants of acylation (k₂). However, values of the dissociation constant (K_S) indicate that plasmin isozyme 1 has a greater binding affinity for these substrates than does isozyme 2. The magnitude of this difference varies with the substrate and is the greatest for those containing analogs of the guanidino moiety of l-arginine.     

Functional role of the activity of ATP-sensitive potassium channels in electrical behavior of hippocampal neurons: experimental and theoretical studies

ATP-sensitive K⁺ (K_ATP) channels are distributed in a variety of cell types, including hippocampal neurons. These channels provide a link between electrical activity of cell membranes and cellular metabolism. The activity of K_ATP channels in hippocampal H19-7 neurons treated with or without short interfering RNAs (siRNAs) directed against Kir6.2 mRNA was investigated in this study. In single-channel recordings, cell exposure to diazoxide (30 μM) significantly prolonged the mean open time of K_ATP channels; however, neither closed-time kinetics nor the single-channel conductance of the channel was altered by this compound. However, in cells transfected with Kir6.2 siRNAs, diazoxide-stimulated activity of K_ATP channels was abolished. Based on single-channel recordings, the activity of K_ATP channels was mathematically constructed in a Markovian manner. The simulated activity of single K_ATP channels was incorporated in a modeled hippocampal neuron to assess how any changes in K_ATP-channel activity affect burst firing of action potentials (APs). The modeled neuron was adopted from the model of Xu and Clancy (2008). Specifically, to mimic the action of diazoxide, the baseline value of open time (τ_bas) of K_ATP channels was arbitrarily elevated, while varying number of active channels (N_O) was set to simulate electrical behavior of Kir6.2 siRNAs-transfected cells. The increase of either N_O or τ_bas depressed membrane excitability of modeled neuron. Fast-slow analysis of AP bursting from this modeled neuron also revealed that the increased K_ATP-channel activity shifted the voltage nullcline in an upward direction, thereby leading to a reduction of the repetitive spike regime. Taken together, it is anticipated that the increased activity of K_ATP channels caused by increasing N_O or τ_bas contributes to or is responsible for burst firing of APs in hippocampal neurons if similar results occur in vivo.     

Probing the role of Valine 185 of the D1 protein in the Photosystem II oxygen evolution

In Photosystem II (PSII), the Mn₄CaO₅-cluster of the active site advances through five sequential oxidation states (S₀ to S₄) before water is oxidized and O₂ is generated. The V185 of the D1 protein has been shown to be an important amino acid in PSII function (Dilbeck et al. Biochemistry 52 (2013) 6824–6833). Here, we have studied its role by making a V185T site-directed mutant in the thermophilic cyanobacterium Thermosynechococcus elongatus. The properties of the V185T-PSII have been compared to those of the WT*3-PSII by using EPR spectroscopy, polarography, thermoluminescence and time-resolved UV–visible absorption spectroscopy. It is shown that the V185 and the chloride binding site very likely interact via the H-bond network linking Tyr_Z and the halide. The V185 contributes to the stabilization of S₂ into the low spin (LS), S?=?1/2, configuration. Indeed, in the V185T mutant a high proportion of S₂ exhibits a high spin (HS), S?=?5/2, configuration. By using bromocresol purple as a dye, a proton release was detected in the S₁Tyr_Z?→?S₂^HSTyr_Z transition in the V185T mutant in contrast to the WT*3-PSII in which there is no proton release in this transition. Instead, in WT*3-PSII, a proton release kinetically much faster than the S₂^LSTyr_Z?→?S₃Tyr_Z transition was observed and we propose that it occurs in the S₂^LSTyr_Z?→?S₂^HSTyr_Z intermediate step before the S₂^HSTyr_Z?→?S₃Tyr_Z transition occurs. The dramatic slowdown of the S₃Tyr_Z?→?S₀Tyr_Z transition in the V185T mutant does not originate from a structural modification of the Mn₄CaO₅ cluster since the spin S?=?3?S₃ EPR signal is not modified in the mutant. More probably, it is indicative of the strong implication of V185 in the tuning of an efficient relaxation processes of the H-bond network and/or of the protein.