heavy metals and plants - a complicated relationship · heavy metals and plants ... iron and zinc...

Heavy Metals and Plants - a complicated relationshipI t t M t ll t i i Pl tImportant Metalloproteins in Plants

Heavy metal hyperaccumulation in the Wild Westmodified from:

Dose-Response principle for heavy metals

Küpper H, Kroneck PMH, 2005, Metal ions Life Sci 2, 31-62

Mechanisms of Metal Uptake in plantsRoot uptake and intracellular distribution in plants

l i d i t t i B iexample: iron and zinc transport in Brassicaceae

root uptake

intracellular distribution

From: Colangelo EP Guerinot ML 2006 CurrOpinPlantBiol9:322 330From: Colangelo EP, Guerinot ML, 2006, CurrOpinPlantBiol9:322-330

4 main familiesP-type ATPases

i diff i f ili (CDF )cation diffusion facilitators (CDF-transporters)ZRT-/IRT-like proteins (ZIP-transporters)Natural resistance associated Macrophage proteins (Nramp-transporters)

Metal sites in photosynthetic proteins

FNRFe3+/2+

Mg2+

A0, A1F

P700*

Mg2+

P680*

Antenna Chl-protein complexes,

FdPhe Fe

FxFA, FB

QA4 h·ν

Chl

main protein:LHCII4 h·ν

t b

PQ

electron

FeQB Cyt b6/f

complex

EETChlChl

ChlChl

Chlcyt b559,cyt c550 PC

transport

P700

p

P680

excitation energy transfer

C

Ca2+ C +/2+

2 H2O O2 + 4 H+

P680

WSC 4 e-Ca2 Cu+/2+

Mn3+/4+

Most abundant magnesium proteins in plants:Light harvesting chlorophyll-protein complexesLight harvesting chlorophyll protein complexes

LHCII structureusually trimersstructure stabilised by Chl

binding via axial ligands on Mg2+

energy transfer depends on 1/r6

From: commons.wikimedia.org

Most important manganese enzyme: Water splitting complex of PSII

)a) structure

Water splitting complex of the photosystem II reaction centre(b) proposed mechanism(b) proposed mechanism

From: McEvoy JP, Brudvig GW, 2006, Chemical Reviews 106, 4455-83

2 of the 4 Mn ions are redox-active (3+/4+) accepting electrons from water and2 of the 4 Mn ions are redox-active ( ), accepting electrons from water and transferring them to P680Ca2+ helps in binding the water

Examples of plant enzymes with iron in the active centreIron is redox-active (Fe3+/Fe2+) usually used for reducing or oxidising metabolites, and ( ) y g gfor transfer of electrons in electron transport chains

CatalaseFerredoxin

Cytochrome c Rieske protein

Biochemistry of iron in iron enzymes

Heme

From: dasher.wustl.edu

Important types of iron-sulfur clusters

From: www.chem.ox.ac.uk

Non-heme iron in the photosystem II reaction centre

From: Utschig LM, Thurnauer MC, 2004, AccChemRes37, 439-47

From: McEvoy JP, Brudvig GW, 2006, Chemical Reviews 106, 4455-83

bound by 4 histidines and 1 glutamate near the stroma surface of the PSIIRCbound by 4 histidines and 1 glutamate near the stroma surface of the PSIIRCguides/helps electrons tunnelling from QA to QB

Catalases, some of the most important

heme-iron enzymes: (a) function and structure

Detoxify hydrogen peroxide (H2O2)Various forms found in all kinds of aerobic organisms from bacteria to plants and animalsI l t h iIron always present as heme-iron

Neurospora crassa catalase1, From: Diaz A et al, 2004, J Mol Biol 342, 971-85

Saccharomyces cerevisae catalase A, From: Maté JM et al, 1999, J Mol Biol 268, 135-49

Catalases, heme-iron enzymes

(b) catalysis(b) catalysis alternative pathways: a) His-mediated mechanism, b) direct mechanism

From: Alfonso-Prieto M et al., 2009, J A Ch S 131 11751 61J. Am. Chem. Soc. 131, 11751-61.

Photosynthesis related Proteins with iron centres5. Photosystem I reaction centre

( ) F i(a) FunctionGeneral

primary charge separation:p y g pspecial pair (=P700, Chl a / Chl a’ heterodimer), releases e- to A0 via A (both Chl a) e- transport via A1 (phylloquinone) and the -520 mV

-580 mV

[4Fe4S]-clusters Fx, FA and FB to the [4Fe4S]-cluster of ferredoxinP700 is re-reduced by plastocyanin

-800 mV

-705 mV

-1000 mV

-800 mV

Function of the 4Fe4S-clusters in PSIRC

+430 mV

Function of the 4Fe4S clusters in PSIRCaccept electrons from the phylloquinones (“A1”)transfer the electrons to ferredoxin

From: Nelson N, Yocum CF, 2006, AnnRevPlantBiol 57, 521-65

Photosynthesis related Proteins with iron centresFerredoxinFerredoxin

Structure and function

usually dimersoluble protein with one [2Fe2S]-cluster per monomertransfers electrons from PSIRC to ferredoxin reductase ( linear electron transport)

C for to the Cyt b6f complex ( cyclic electron transport)

From: www.fli-leibniz.de with reference to data of Bes MT, Parisini E, Inda LA, Saraiva LM, Peleato ML, Sheldrick GM, 1999, Structure, 15;7(10):1201-11

Selected enzymes with copper in the active centreCopper is redox-active (Cu2+/Cu+) usually used for reducing or oxidising metabolites, pp ( ) y g gand for transfer of electrons in electron transport chains

• Plastocyanin

•Cytochrome c oxidase 3 Cu (binuclear site and mononuclear(binuclear site and mononuclear site)

•SOD = Superoxide dismutase

Copper delivery inside cellular compartments

f hi bl th i th 1990’ i i 2000 6 i 2

From: OHalloran TV, Culotta VC, 2000, JBC275, 25057-60

fashionable theme since the 1990’s, numerous reviews since 2000, 6 reviews on 2 chaperones by Rosenzweig and O’Halloran, with mostly identical figures in 4 reviews 2000+2001confusing large number of names for homologous proteins in different organismsconfusing large number of names for homologous proteins in different organismsREALITY: just 3 really different (non-homologous) Cu-chaperones are well known, some more proteins are postulated to be Cu-chaperones

Metallothioneins: storage and transport proteins

From: Prohaska JR, Gybina AA, 2004,

JNutrition134, 1003-6

• MTs of type I und II bind Cu+ with high affinity and seem to be involved in its detoxification.

• BUT: Main role of MTs in plants seems to be• BUT: Main role of MTs in plants seems to be Metal-distribution during the normal (non-stressed) metabolism, while in animals they are more involved in detoxification and also (mainly?)more involved in detoxification and also (mainly?) bind Zn2+

Copper delivery to the Golgi and thylakoid:ATX1 = HAH1 = ATOX1 = CopZ ≈ CCH

(a) occurrence

From: OHalloran TV Culotta VC 2000 JBC275 25057 60From: OHalloran TV, Culotta VC, 2000, JBC275, 25057-60

ATX1 found in yeast originally as a gene involved in protection against oxidative damage; human homologue: HAH1 = ATOX1gbacterial homologue: CopZcyanobacterial and homologues: Atx1similar to plant CCH

Copper delivery to the Golgi and thylakoid:ATX1 = HAH1 = ATOX1 = CopZ

(b) function in bacteria, cyanobacteria+plants

CopZ in non-photosynthetic bacteria donates Cu to CopY transcription factor? Or Cu delivery to Cu-CopY transcription factor? Or Cu delivery to Cuefflux transport ATPase CopB or copper influx ATPase CopA?Atx1 found to specifically shuttle copper to anAtx1 found to specifically shuttle copper to an intracellular CPx-type copper ATPase the thylakoid in cyanobacteria+plantsCtaA+PacS ATPases deliver Cu for plastocyanine + p yCyt c6 across thylakoid membrane

From: Borrelly GPM et al., 2004, BiochemJ378, 293-7

Copper delivery to the Golgi and thylakoid:ATX1 = Atx1 = HAH1 = ATOX1 = CopZ

(b) t t(b) structure

From: Rosenzweig AC, OHalloran TV, 2000, CurrOpinChemBiol4, 140-7g p

Ca. 70 residuesβαββαβ fold with two alpha helices superimposed on a 4 stranded beta sheetβαββαβ-fold with two alpha-helices superimposed on a 4-stranded beta-sheet

Copper delivery to the Golgi and thylakoid:ATX1 = HAH1 = ATOX1 = CopZ = Atx1

( ) C bi di i b t i l b t i l l t i(c) Cu-binding in bacterial+cyanobacterial+plant versionAtx1

(Cyanob.)

F B ll GPM t l 2004 Bi h J378 293 7

Atx1 binds a single Cu(I) ion like ATX1Atx1: like in the yeast+animal proteins Cu binding via two Cys in the sequence

From: Borrelly GPM, et al., 2004, BiochemJ378, 293-7

Atx1: like in the yeast+animal proteins, Cu-binding via two Cys in the sequence MT/HCXXC, BUT additional histidine61 from loop 5the additional histidine shifts Atx1 binding affinity towards CtaA by reducing affinity for PacS trafficking of Cu(I) from one CtaA to the PacSfor PacS trafficking of Cu(I) from one CtaA to the PacSother features like in yeast+animal proteins

COPPER CHAPERONE =

CCH

F Pil M t l 2006From: Pilon M et al., 2006, CurrOpinPlantBiol9, 256-63

Interacts with HMA5 in Arabidopsis t i C i th t l d d li i it t t G l i lsequestering Cu in the cytoplasms and delivering it to post-Golgy vesicles

expression upregulated by Cu-stress and during senescence, the latter because it is involved in copper delivery to ethylene receptorsexpression around vascullar tissue and protein found in phloemexpression around vascullar tissue and protein found in phloemtransport of Cu by the carboxyterminal domain through plasmodesmata to non-nucleated cells like sieve elements, symplastic intercellular Cu-transport pathway?

Copper delivery to cytosolic SODCCS = LYS7 = SOD4

f ti dfunction and occurrence

From: OHalloran TV, Culotta VC, 2000, JBC275, 25057-60

CCS delivers Cu to Cu/Zn requiring SOD1 in yeast and to CSD1 and CSD3 in plantsCCS = yeast LYS7 = human homologue SOD4


(b) t t(b) structure

From: Rosenzweig AC, OHalloran TV, 2000, CurrOpinChemBiol4, 140-7

dimer? CCS folds into three functionally different protein domainsN terminal domain I has striking homology to ATX1 incl the MXCXXC Cu binding

g p

N-terminal domain I has striking homology to ATX1, incl. the MXCXXC Cu-binding siteCentral domain II is homologous to its target, SOD1 (mutation causes SOD-activity in human SOD4!) and physically interacts with thatin human SOD4!) and physically interacts with thatC-terminal domain III (30 residues) is extremely crucial for SOD1 activation, highly conserved, CXC Cu-binding motif in loop before α-helix


( ) h i(c) mechanism

From: Rosenzweig AC, 2002, ChemBiol9, 673-7

Domain I likely recruits copperDomain II facilitates recognition of SOD1D i III b bl di t d li b bl t th ith d i IDomain III probably mediates copper delivery, probably together with domain I

One of the most important copper enzymes: Superoxide dismutase (SOD), in plants a Cu/Zn enzyme

( ) f ti(a) function

From: Foyer CH et al., 1994, PlantCellEnvi17 507-23y , , _

Present in all aerobic organisms, particularly important in photosynthetic organismsDetoxifies superoxide that was generated e.g. by photosynthesis or respiration

One of the most important copper enzymes: Superoxide dismutase (SOD), in plants a Cu/Zn enzyme

(b) t tDimer of two identical subunits, in crystals 2 dimers together

(b) structure

each subunit consists of:- 8 anti-parallel β-strands forming a flattened

cylinder, 3 t l l- 3 external loops

1 Cys-Cys disulfide bond stabilises loops1 Cu2+ and 1 Zn2+ per subunitCu2+ bound by 4 His Zn2+ by 3 His + 1 AspartateCu2+ bound by 4 His, Zn2+ by 3 His + 1 AspartateHis-63 bridges Cu2+ and Zn2+

: KD of copper: 10-15 M

CuZn

Spinach SOD, From: Kitagawa Y et al., 1991, J Biochem 109, 477-85, images generated with Jena 3D viewer

Selected plant enzymes with zink in the active centre

2Zn2+ is redox-indert functions in proteins as stabiliser of the structure (e.g. Zinc fingers for gene expression regulation) or as a Lewis-base catalyst (e.g. carboanhydrases)

Carboanhydrase

Zink finger motiveZink finger motive

Tyrosin-Phosphatase

Photosynthesis related Enzymes with metal centresCO2 delivery: Cd- and Zn- carboanhydrases2 y y

(a1) functionfunction of carboanhydrases (from:

bi l ih /b k h lf/b f i?b k & A1199)www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=stryer&part=A1199)

Convert carbon dioxide to bicarbonate and vice versaPresent in all aquatic photosynthetic organisms as part of the Carbon Concentrating Mechanism (CCM)

HCO P

HCO3- HCO3

- CO2

HCO3- Pump Carboanhydrase

Photosynthesis

Present in most respiratory organisms (incl. animals like us!) for removing CO2 from th b d b h l ti

Plant cell

the body by exhalation

HCOCOCOCarboanhydrase

RespirationHCO3-CO2CO2

Respiration

Animal cell


(a2) reaction mechanism

By lowering the pKa of water from 15.7 to 7, the binding of water to Zinc facilitates the release of a proton, which generates a hydroxide ion.

C fCarbon dioxide binds to the active site of the enzyme and is positioned to react with the hydroxide ion.

The hydroxide ion attacks the carbon dioxide, converting it into a bicarbonate ionion.

The catalytic site is regenerated with the release of the bicarbonate ion and the

Reaction mechanism of Zn-carboanhydrases (from: www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=stryer&part=A1199)release of the bicarbonate ion and the

binding of another molecule of water.www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book stryer&part A1199)


(b) comparison of Cd- and Zn-Carboanhydrases

Cd CA is much larger than Zn CACd-CA is much larger than Zn-CA

Cd-CA can bind both Cd and Zn. Activity with Zn slightly, but Activity with Cd much higher than in regular Zn-Carboanhydraseshigher than in regular Zn Carboanhydrases.

Porphyridium Zn-CA (Mitsuhashi S et al., 2000, JBC 275, 5521-6)

Thalassiosira Cd-Carboanhydrase (Xu et al., 2008, Nature 452, 56-61)

From: Lane and Morel, 2000, PNAS Vol. 97


(b1) structure and properties of a typical Zn-CA

Z CA i h di• Zn-CA is a homodimer

• each monomer consists of and α/β-domain and 3 α-helices

•Zn2+ is coordinated by 2 Cys, 1 Asp and 1 His

Zn-Carboanhydrase from the marine red alga Porphyridium (Mitsuhashi S et al., 2000, JBC 275, 5521-6)


(b2) Properties and Structure of the Cd-Carboanhydrase

•Cd-CA has 7 α-helices and 9 β-sheats,

•Cd is at the lower End of a funnel-like substrate binding pocket

Cd2+ i b d b 2 C d 1 Hi•Cd2+ is bound by 2x Cys and 1x His, plus 1x Water ( tetrahedral coordination).

Cd-Carboanhydrase from the marine diatom Thalassiosira weissflogii, Xu et al., 2008, Nature 452, pp 56-61

Examples of Nickel complexes in proteinscomplexes in proteins

Characteristics

Nickel is usually bound by nitrogen (mainly histidine),

l h ( t i ) dsulphur (cysteine) and oxygen ligandsusually 5 (4-6) ligands

From: Carrington PE et al, 2002, EnvHealthPers110, 705-

Best known (and most important?) Ni-enzyme: Ureasea) Function and occurrence)

Catalyses the decomposition of urea into carbon dioxide and ammoniaIn most organisms (plants, fungi, bacteria)Very important for metabolism: urea toxicity is one of the main mechanisms of damage caused by nickel deficiency in plantsVery specific for urea as a substrateRather fast: turnover rate kcat around 3,500 s-1

Ureaseb) Active site

From: Lee WZ et al., 2008, DaltonTrans, 2538-41a o a s, 538

t Ni2+ itwo Ni2+ ionsone Ni2+ bound by three fixed ligands (2 His-N and 1 Lys-O) and one waterthe other Ni2+ bound by four fixed ligands (2 His-N, 1 Asp-O and 1 Lys-O) and 1 waterwaterthe two nickels are bridged by a water molecule

Urease c) Mechanismc) Mechanism

StepsNickel1 binds urea at the oxygenNickel 2 binds

twater tetrahedral intermediateafter cleavage ofafter cleavage of the C-N bond, carbamic acid is bound to thebound to the nickels

From: Karplus PA, Pearson MA, Hausinger RP, 1997,

Acc Chem Res 30 330-37Acc Chem Res 30, 330-37,modified by

Estiu G, Merz KM, 2004, JACS126, 11832-42

All slides of my lectures can be downloaded from my workgroup homepage

www.uni-konstanz.de Department of Biology Workgroups Küpper lab,

or directly

http://www uni-konstanz de/FuF/Bio/kuepper/Homepage/AG Kuepper Homepage htmlhttp://www.uni-konstanz.de/FuF/Bio/kuepper/Homepage/AG_Kuepper_Homepage.html

heavy metals and plants - a complicated relationship · heavy metals and plants ... iron and zinc...

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