Effect of lanthanides on oxidation of Mn.pdf

Journal of Inorganic Biochemistry 244(2023)112237Available online 23 April 20230162-0134/2023 Elsevier Inc.All rights reserved.Effect of lanthanides on oxidation of Mn2+cations via a high-affinity Mn-binding site in photosystem II membranes E.R.Lovyagina,O.G.Luneva,A.V.Loktyushkin,B.K.Semin*Department of Biophysics,Faculty of Biology,Lomonosov Moscow State University,Moscow 119234,Russia A R T I C L E I N F O Keywords:Photosystem II Lanthanides High-affinity Mn-binding site Mn2+oxidation Oxygen-evolving complex Calcium A B S T R A C T Lanthanide cations(La3+and Tb3+)bind to the Ca-binding site of the oxygen-evolving complex in Ca-depleted PSII membranes and irreversibly inhibit the oxygen evolution.n the other hand,EPR measurement of Mn2+concentration in buffer revealed that lanthanide cations inhibit the light-dependent oxidation of Mn2+cations via the high-affinity Mn-binding site in Mn-depleted PSII membranes,which suggests that they bind to and inhibit the high-affinity Mn-binding site of the oxygen-evolving complex.The inhibition is irreversible,bound Ln3+cation could not be washed out from the sample.Calcium ion inhibits oxidation of Mn2+(5 M)at very high concentration(tens mM)and the inhibition is reversible.In this work we measured the reduction rate of exogenic electron acceptor 2,6-dichlorophenolindophenol during the oxidation of Mn2+cations in the Ca-depleted PSII and in the Ca-depleted PSII treated with lanthanides after extraction of Mn cluster from these preparations.We found that irreversible binding of the lanthanide cation to the Ca-binding site in the Ca-depleted PSII membranes leads to a partial inhibition of the high-affinity Mn-binding site.1.Introduction The catalytic center of the oxygen-evolving complex(OEC)in the oxygenic photosynthetic organisms carries out one of the most impor-tant bioenergetic reactions-water oxidation and molecular oxygen synthesis.The catalytic center consists of 4 Mn and 1 Ca2+cations connected to each other by 5 oxygen bridges 1,2.Metal cluster have four molecules of water,two of which are bound to Mn4 cation and the other two bound to Ca2+cation.One of the Ca2+-bound water molecules is presumably a substrate molecule,which is involved in formation of molecular oxygen 3,4 although the mechanism of Ca2+function in water oxidation might be different.For example,Ca2+can be respon-sible for an“adjustment”of the redox potential of a manganese cluster 57.The Ca2+cation can be extracted from OEC of photosystem II(PSII)with high ionic strength medium(12 M NaCl)8 or low pH citrate buffer 9.This procedure is accompanied by a loss of oxygen-evolving activity:the residual rate of oxygen evolution is about 12%.The activity of Ca-depleted PSII membranes(PSII(Ca)can be restored up to 70%by addition of Ca2+in high concentrations(1030 mM)10.A number of metal cations have been investigated as a substitute for Ca2+in OEC 11.However,the only metal ion which could functionally substitute Ca2+is Sr2+12.The binding of some other metal ions,including lanthanides(Ln3+)13,14 and Cd2+15,to the Ca-binding site is competitive with Ca2+.Lanthanides irreversibly bind to the Ca-binding site in intact membrane preparations of the PSII,displacing Ca2+cation from the OEC 13,as well as to free Ca-binding site in Ca-depleted PSII membranes 14.However,the mechanism of interaction of lanthanides cations with PSII membranes might be more complex than simply binding to the Ca-binding site of the OEC.The essence is that the PSII contains,in addition to Ca2+cation,4 Mn cations,which can be extracted from OEC together with Ca2+cation and extrinsic proteins.The Mn-depleted PSII samples(PSII(Mn)contain single Mn-binding site 16,17,which binds Mn2+cations with high efficiency.This high-affinity site(HAS)is located at the position denoted by Mn4 1 in the native crystal structure.The HAS was assigned to axial ligands Asp170 and Glu333 in the D1 polypeptide of the native PSII membranes 17.This site can presumably bind Fe2+Abbreviations:Chl,chlorophyll;DCPIP,2,6-dichlorophenolindophenol;DPC,1,5-diphenylcarbazide;HAS,high-affinity Mn-binding site;Ln3+,lanthanide cation;MES,2-(N-morpholino)-ethanesulfonic acid;OEC,oxygen-evolving complex;PSII,photosystem II;PSII(Ca),Ca2+-depleted PSII membranes;PSII(Mn),Mn-depleted PSII membranes;RC,reaction center;SD,standard deviation;Tris,tris(hydroxymethyl)aminomethane.*Corresponding author.E-mail address:seminbiophys.msu.ru(B.K.Semin).Contents lists available at ScienceDirect Journal of Inorganic Biochemistry journal homepage: https:/doi.org/10.1016/j.jinorgbio.2023.112237 Received 11 February 2023;Received in revised form 4 April 2023;Accepted 19 April 2023 Journal of Inorganic Biochemistry 244(2023)1122372cations with a comparable to Mn2+efficiency 18.Recently,a detailed study of the photoactivation process in cyanobacterial membranes led the authors to a conclusion that there is a second Mn-binding site in the apo-PSII membranes 19.This was confirmed by Mino and Asada(2022)using the pulsed electronelectron double resonance 20.The authors suggested that the position between Asp170 and Glu189 in the D1 polypeptide is a good candidate for the initial high-affinity site for photoactivation.In our previous work 21,we found that La3+and Tb3+cations effectively inhibited the reduction of artificial electron acceptor 2,6-dichlorophenolindophenol(DCPIP)supported by oxidation of Mn2+cations at the Mn-binding HAS in the Mn-depleted PSII membranes.The Ki values are equal to 2 M for Tb3+and 8 M for La3+,which is close to the concentration of Fe2+cations(3 M)that blocks the oxidation of Mn2+cations through the HAS 18.In contrast,the Ki inhibition of the Ca-binding site in PSII(Ca)membranes(200 M)is much greater 14.These facts suggest that the lanthanide cations inhibit the oxidation of Mn2+cations bound to the HAS,blocking the binding of Mn2+cations,which is similar to the Mn2+oxidation blocking by iron cations 18.In this work,we continued to study the mechanism of Ln3+cations inter-action with the HAS by EPR method.The obtained results confirm the fact of highly effective binding of lanthanide cations to the Mn-binding HAS in the PSII(Mn)membrane preparations.We found that irre-versible binding of the lanthanide cation to the Ca-binding site in the Ca-depleted PSII membranes leads to a partial inhibition of the high-affinity Mn-binding site.2.Materials and methods 2.1.PSII preparations The PSII-enriched membrane fragments(BBY-type)were prepared from market spinach leaves following protocol of Ghanotakis and Bab-cock(1983)22.The O2-evolving activity of the native PSII mem-branes,measured polarographically,ranged from 450 to 550 mol O2 mg Chl 1 h 1,when 0.2 mM 2,6-dichloro-p-benzoquinone(Sigma-Aldrich)was used as an artificial electron acceptor.The preparations were stored at 80 C in buffer A,containing 15 mM NaCl,400 mM sucrose,and 50 mM MES/NaOH(pH 6.5).Chlorophyll(Chl)concen-trations were determined in 80%acetone,according to the method of Porra et al.(1989)23.Samples were thawed in the dark at 5 C in a refrigerator for 70 min before treatment or measurement.Concentration of PSII reaction centers(RC)is presented in M on the basis of 250 molecules of Chl/RC of PSII sample 24,25.2.2.Measurement of photoinduced DCPIP reduction Electron transport activity in PSII preparations was measured as the rate of the exogenous electron acceptor DCPIP photoreduction(calcu-lated for the deprotonated form of DCPIP)using a Specord UV-VIS spectrophotometer(Carl Zeiss Jena,Germany)in cuvettes with 1 cm optical path length.The XBDROY light diodes(Cree Inc.,United States)with a maximum of 450 nm were used as the exciting light source to provide a saturating light intensity of 1800 E m 2 s 1.The cut-off excitation orange glass filter OS-14 was installed in front of the photo-multiplier tube of the spectrophotometer.Photoinduced changes in DCPIP optical density were recorded at wavelength of 600 nm.Reduc-tion rate of DCPIP was determined from these changes using the molar extinction coefficient,=21.8 mM 1 cm 1 26.2.3.Ca2+extraction from PSII membranes The PSII(Ca)membranes were prepared by incubating PSII mem-branes in buffer,containing 2 M NaCl,0.4 M sucrose,and 25 mM MES(pH 6.5)8.The preparations were incubated in the buffer for 15 min at room temperature under room fluorescent light(45 E m 2 s 1).The resulting material was then washed twice with buffer A.The obtained PSII(Ca)membranes also lack the PsbQ and PsbP extrinsic proteins,which shield OEC from attack by exogenic factors.2.4.Mn extraction from PSII membranes Tris treatment.Manganese-depletion was accomplished by incubating thawed PSII membranes(0.5 mg Chl/ml)in 0.8 M Tris-HCl buffer(pH 8.5)for 15 min in the light at room temperature(22 C).The membranes were then pelleted by centrifugation in an Eppendorf centrifuge 5415R(16,100 g 3 min),washed 3 times with buffer A to remove non-specific Mn2+,and finally re-suspended in buffer A.These membranes do not contain any extrinsic proteins(including PsbO polypeptide),Ca2+ion,and the Mn catalytic cluster.Residual Mn content in Mn-depleted PSII membranes after Tris treatment was 0.3 0.1 Mn/RC(here and below:mean SD).Hydroxylamine treatment.Manganese-depletion was also accom-plished by incubating thawed Ca-depleted PSII membranes(0.5 mg Chl/ml)for 10 min at 46 C in the dark with 1 mM hydroxylamine in buffer A 27.The membranes were then pelleted by centrifugation and washed 3 times with buffer A.These membranes do not contain two of the extrinsic proteins(PsbP and PsbQ),Ca2+ion,and the Mn cluster.Residual Mn content in Mn-depleted PSII membranes after hydroxyl-amine treatment was 0.4 0.1 Mn/RC.2.5.EPR measurements EPR was used to detect the light-dependent oxidation of exogenous Mn2+cations(MnCl24H2O)in different PSII preparations by measuring six-line spectra.The PSII membranes(1 mg Chl/ml)were put into a flat-type quartz EPR cell(70 l)in buffer A and illuminated directly in the cavity of an EPR spectrometer(RE1307,USSR)with XBDROY light di-odes(Cree Inc.,United States)with a maximum in the region of 450 nm(1800 E m 2 s 1).The EPR settings were:microwave power 20 mW,time constant 0.1 s,sweep time 100 s.All experiments we performed at 20 C.Each characteristic spectrum presented in the figures is an average of 5 replicates.2.6.Determination of the Mn content in PSII samples The amount of manganese cations in OEC was controlled using the method of colorimetric determination of the Mn2+concentration 28 with minor changes described earlier 29.A suspension of PSII prepa-rations in buffer A(chlorophyll concentration 100 g/ml)was incubated with 50 mM CaCl2 at 5 C in the dark for 2 min to remove nonspecifically bound Mn ions.Then,the PSII membranes were precipitated and the pellet was resuspended in 0.6 N HCl in a 1:9 volume ratio to extract functional Mn from the OEC.After 10-fold dilution with distilled water,the suspension was centrifuged(16,100 g 3 min)and the supernatant was filtered through a 0.2 m nylon membrane(Pall Life Sciences,Ann Arbor,USA).40 l of 2 M NaOH,40 l of a solution of 3,3,5,5-tetra-methylbenzidine(100 mg in 100 ml of 0.1 M HCl)and 40 l of 5.3 M H3PO4 were successively added to 0.9 ml of the filtrate.To determine the concentration of Mn2+,absorption was recorded at 450 nm(extinction coefficient 34 mM 1 cm 1)30.When calculating the PSII RC con-centration,the number of chlorophyll molecules per RC was assumed to be 250 24,25.3.Results and discussion In previous work,we have shown that La3+and Tb3+inhibit the reduction of the exogenous electron acceptor DCPIP in PSII(Mn)preparations.As an electron donor,we used Mn2+cations(2 M)in combination with hydrogen peroxide(3 mM).This donor system Mn2+H2O2 donates electron only via the Mn-binding HAS 3133.The Mn2+cations bound to the HAS are oxidized by the tyrosine radical YZ E.R.Lovyagina et al.Journal of Inorganic Biochemistry 244(2023)112237334.Oxidized Mn2+cations are reduced by hydrogen peroxide,which significantly increases the rate of electron transport to DCPIP.In this work,to simplify the study system,we used the EPR method to inves-tigate the Mn2+oxidation reaction and the effect of Ln3+cations on this process.In this case,it is not necessary to use a DCPIP acceptor and hydrogen peroxide in the experiment,which eliminates the possibility of their participation in some side reactions.It is known that Mn2+cations have a six-line EPR spectrum in buffer A(without PSII membranes)(Fig.1a)or in the suspension of PSII membranes(Fig.1b),which dis-appears when they are oxidized(Fig.1b,under illumination).This EPR property of the Mn cation is often used,for example,in estimations of the Mn content of PSII preparations.Indeed,when PSII(Mn)mem-branes are illuminated in the presence of exogenous Mn2+cations,its EPR signal disappears that show their oxidation on the donor side of the photosystem.When the light is turned off,the EPR signal of Mn2+ap-pears(Fig.1b,after illumination)due to the reduction of Mn3+cations as a result of charge recombination reactions.Photooxidation of Mn2+cation does not require Ca2+in the medium 3437.However,in the case of photoactivation,Ca2+appears to be involved in the oxidation of Mn2+.The absence of Ca2+in intermediate A of the photoactivation process decreases the rate of electron donation to YZ 38 and increases the back reaction of intermediate B to A 19.Calcium must be present to accumulate intermediate B 3940 that could be assigned as a Mn3+-(OH)-Ca2+or Mn3+-(O2)-Ca2+oxide ion species 41.An addition of Ca2+to the buffer with PSII(Mn)membranes suppresses Mn2+oxidation(Fig.1c).This could be explained by following mechanisms.1)It is known that Ca2+and Mn2+compete for each others binding sites 42,43.Huge difference in con-centration of Mn2+(5 M)and Ca2+(30 mM)suggests a competitive mechanism of calcium inhibition.2)The Ca2+is involved in photo-reconstruction of Mn cluster associated with oxidation of Mn2+cations 43.Chen et al.have shown that Ca2+inhibits the nonfunctional Mn2+oxidation 44.It is more probable that both mechanisms are switched on in process of inhibition of Mn2+oxidation by calcium.Oxidation of Mn2+takes place at very low concentration(5 M,Fig.1b),which in-dicates a high affinity of the binding site.When Ca2+is added at a very Fig.1.Effect of La3+and Ca2+cations on oxidation of Mn2+cations on the donor side of PSII(Mn)membranes.a)EPR spectrum of Mn2+cations(5 M)in buffer A.b)Oxidation of Mn2+cations(5 M)in the presence of PSII(Mn)membranes(1 mg Chl/ml)under illumination(ratio RC:Mn2+1:1.25 in M).c)PSII(Mn)membranes(1 mg Chl/ml)were incu-bated for 10 min at 4 C with 30 mM CaCl2,then Mn2+(5 M)was added and the EPR spectra were measured.d)PSII(Mn)membranes(60 g Ch/ml)were incubated with La3+(2 mM)for 3 min in room light.The membranes were then precipitated by centrifugation,resuspended in buffer A(1 mg Chl/ml),Mn2+(5 M)was added and EPR spectra were measured.The RC:La3+concentration ratio is 1:500.e)PSII(Mn)membranes(