Evidences for structural basis of altered ascorbateperoxidase activity in cadmium-stressed rice plantsexposed to jasmonate

Indra Singh • Kavita Shah

Received: 27 November 2013 / Accepted: 5 January 2014 / Published online: 18 January 2014

� Springer Science+Business Media New York 2014

Abstract Binding interactions of cadmium (Cd) with

rice ascorbate peroxidase (OsAPX) in presence or

absence of jasmonate was examined in-silico. OsAPX is

a 250 amino acid long protein with 90 % sequence

similarity to soybean-APX. The 3D model of OsAPX

obtained by homology modeling using soybean

APX (PDBID:1OAF) as template was associated

with -15975.85 kJ/mol energy, 100 % residues in

favoured region, verify score of 0.85, ERRAT score

89.625 and a negative ProSA graph, suggesting OsAPX

model to be of good quality, robust and reliable which was

submitted with Protein Model Database with PMDBID:

PM0078091. The rice ascorbate peroxidase ascorbate

[OsAPX–Asc] complex had a substrate binding cavity

involving residues at position 30KSCAPL35, 167RCH169

and 172R wherein ascorbate accommodated via three

H-bonds involving 30Lys at the c-edge of heme. 169His

served as a bridge between heme-porphyrin of OsAPX

and ascorbate creating a charge relay system. Cd bound

in [OsAPX–Asc–Cd] complex at 29EKSCAPL35, a site

similar to ascorbate binding site. The binding of Cd

caused breaking of 169His bridge shifting the protein

conformation. Cadmium exhibited four electrostatic

interactions via 29Glu of OsAPX backbone. Docking of

[OsAPX–Asc] with jasmonic acid (JA) resulted in

[OsAPX–Asc–JA] complex where 4—H-bonds held JA

to OsAPX in a cavity at c-edge on the distal side of

heme. The binding of [OsAPX–Asc–JA] to Cd show the

metal to bind at a position other than that involved in

binding of OsAPX with Cd alone. Results indicate that

Cd does not replace iron or ascorbate or JA but binds to

OsAPX on the surface at a separate site electrostatically.

In presence of JA the interactions involved in formation

of [OsAPXAsc] are restored which is otherwise altered

by the presence of Cd. The formation and reformation of

H-bond take place between the [OsAPX–Asc] and Cd/

JA. It is the interaction between heme and ascorbate

which is modulated differently in presence of Cd/JA. In

absence of JA, Cd-binds to the [OsAPX–Asc] complex

at the proximal end of APX near Asc-binding site,

whereas in presence of JA, Cd-binds on the opposite site

of the Asc-binding site involving 30Lys and 29Glu

residues. In-silico binding studies well correlate with the

wet-lab results where exogenous application of JA

increased the activity of OsAPX in rice grown under Cd-

stress. Therefore it is concluded that the activity of

OsAPX in rice roots and shoots are compromised under

Cd-stress alone.

Keywords Ascorbate peroxidase � Cadmium �Homology modelling � Jasmonate � Rice

All the authors have contributed equally to this work.

I. Singh

Bioinformatics Division, Mahila Mahavidyalaya, Banaras

Hindu University, Varanasi 221005, India

K. Shah (&)

Institute of Environment and Sustainable Development,

Banaras Hindu University, Varanasi 221005, India

e-mail: kavitashah@bhu.ac.in; kshah.iesdbhu@sify.com

123

Biometals (2014) 27:247–263

DOI 10.1007/s10534-014-9705-z

Introduction

It is known that ascorbate peroxidase (APX, EC

1.11.1.11) are class I heme containing enzymes which

catalyze the H2O2-dependent oxidation of ascorbate

(Raven 2003) and have specific structural, functional

and substrate binding properties. It is the substrate

binding properties of APX that places it at an

important interface between peroxidases. The close

sequence identity to class I cytochrome-C-oxidase

coupled with its bifunctional substrate specificity that

mimics both class II and III peroxidases, qualifies

APX as a useful enzyme for enzyme-substrate binding

studies.

Cadmium, a widespread non-essential toxic heavy

metal, is well known to affect various cellular

processes through membrane damage, disruption of

electron transport, enzyme inhibition/activation and

DNA alteration (Shah et al. 2001a, b). Cadmium

toxicity is a consequence of oxidative stress, caused by

the stimulation, formation and accumulation of free

oxygen radicals like H2O2, OH˙, O2

-, etc. (Sanita’ di

Toppi and Gabbrielli 1999; Singh and Shah 2012;

Shah et al. 2013) and altered activity of various

antioxidant enzymes including APX (Hegedus et al.

2001; Shah et al. 2001a, b, 2013). Ascorbate perox-

idase form an important component of Halliwell-

Asada pathway which is well known to be involved in

combating/neutralizing/avoiding the effects of abi-

otic/biotic stresses including heavy metal stress in

plants (Nahakpam and Shah 2011). Reports on the

response of APX expression to stress conditions

indicate the importance of APX activity in controlling

the reactive oxygen species (ROS) concentration in

the intracellular signaling (Shah and Nahakpam 2012;

Rai et al. 2012, 2013).

Jasmonates (JA) have also been proved to regulate

the antioxidant system in plants cells (Wang

et al.1999). Exogenous application of JAs are shown

to elicit defense related specific responses to stress,

thereby increasing resistance of plant towards that

stress (Ding et al. 2001; Wahid et al. 2007). Jasmo-

nates are also reported to counteract the effect of

heavy metal Cd in plants (Moussa and EL-Gamal

2010; Junyu et al. 2010; Belkhadi et al. 2011;

Metwally et al.2003; Keramat et al.2009), however

the mechanism involved is yet unclear. In the wet-lab

studies we observed that exogenous application of

jasmonic acid suppressed the effect of cadmium (Cd)

induced oxidative stress in rice (data communicated

elsewhere).

Rice (Oryza sativa L.) is a globally important crop

with completely sequenced genome (Ohyanagi et al.

2006). It is expected that the 3-D structures of

important rice proteins would now be available in

the protein databases online. Surprisingly, no crystal-

lographic structure could be obtained or is reported for

rice APX protein.

Here we attempt to model the 3-D structure of rice-

APX (OsAPX) by homology and to explore its binding

with substrate (ascorbate) and Cd-metal in silico. A

detailed study of interactions between enzyme-sub-

strate complex and Cd and the alterations in the bonds

formed in presence of JA in silico is reported for the

first time. The results presented here would be helpful

in understanding the mechanism as to how APX

interacts with Cd and/or JA, altering thereby its

activity to mitigate effect of Cd in presence of JA in

rice.

Materials and methods

In silico studies

All computations and simulations were carried out on

an Intel dual core based Microsoft Windows XP

professional workstation.

Sequence retrieval, template search, secondary

structure prediction and sequence alignment

A search for rice APX at National Centre for

Biotechnology Information (NCBI) database revealed

the presence of 125 entries. Amino acid sequence of

ascorbate peroxidase from rice (OsAPX, accession no.

BAA08264.1) was retrieved from the protein

sequence database hosted in the NCBI (http://www.

ncbi.nlm.nih.gov). Using the same source, the con-

served domain search on the query sequence, simi-

larity and identity of the sequence with the soybean-

APX sequence was performed. Sequence of rice APX

was analyzed for conserved domain at INTERPRO

(Attwood et al. 2001) and gene ontology studies were

carried out.

To create a model of the OsAPX, at first a BLAST

search for protein with similar sequence was

248 Biometals (2014) 27:247–263

123

performed using the amino acid sequence of OsAPX

retrieved previously as query. BLASTp (Altschul et al.

1990) was used to identify and retrieve homologous

3D structures by searching the structural database of

protein sequence in the protein databank (PDB)

(Bernstein et al. 1977) (http://www.rcsb.org). The

soybean-APX sequence was identified to have highest

identity among proteins in PDB. Secondary structure

prediction of OsAPX at PROCHECK and sequence

alignment was done using BLAST2 (employing soy-

bean-APX secondary structure corresponding to

PDBID: 1OAF) (Sharp et al. 2003).

Retrieval of the ligand structures (heme, ascorbate,

cadmium and jasmonate)

The 3-D structure of organic compounds heme

(CID:53627695), ascorbate (CID:54670067), cad-

mium (CID:23973), and JA (CID:5281166) were

retrieved at PubChem database (http://pubchem.ncbi.

nlm.nih.gov/). IUPAC name and molecular weight of

compounds are summarized in Table 1. Each of these

molecules were retrieved i n sdf.file format from

PubChem and converted to pdb.file format using

Molegro software (http://www.molegro.com). Files

obtained were used for visualizing 3D structures and

for subsequent docking studies.

Homology modeling, model optimization

and validation

The three dimensional model of APX was predicted by

homology modeling. For modeling of the OsAPX, the

method involving the satisfaction of the spatial

restraints incorporated in the program Modeller 9v

of Accelrys Discovery Studio (DS) was used. A set of

25 models for OsAPX were created by Accelrys-DS

(http://accelrys.com/products/discovery-studio) using

the crystal structure of soybean-APX (PDBID: 1OAF)

as template. The 25 models at various refinement level

and library schedules obtained were verified by DS

verify protein tool. The best model was selected for

energy minimization based on dope score. Energy

were minimized by using smart minimize of DS with

maximum 200 steps with steepest descent technique.

The best OsAPX model obtained after energy mini-

mization was validated using the program PRO-

CHECK (http://nihserver.mbi.ucla.edu/SAVES_3/

saves.php). The stereochemical quality of model and

protein backbone was inspected by Ramachandran

plot analysis (Laskowski et al. 2005). The quality of

the model were also assessed at Verify 3D (Luthy et al.

1992), ERRAT (Colovos and Yeates 1993) and proSA

(Wiederstein and Sippl 2007). The model were sub-

mitted at Protein Model Data Base online and PMDB

ID were obtained.

Molecular docking studies of OsAPX

with ascorbate, cadmium and JA alone

or in combination

The three dimensional structure of OsAPX obtained

above was used for docking studies. Stepwise protein–

ligand dockings were performed using Molegro Vir-

tual Docker (http://www.molegro.com/products.php).

The following docking experiments were carried out

and analyzed (i) OsAPX protein backbone with heme

cofactor containing Fe at its centre to obtain APX-

apoenzyme (ii) OsAPX-apoenzyme with substrate

ascorbate, to obtain [OsAPX–Asc] complex (iii)

Table 1 Summary of the ligands used for docking with rice Os-APX as obtained at Pubchem database online (http://pubchem.ncbi.

nlm.nih.gov/)

Ligand IUPAC name Chemical identification number (CID) Molecular weight

Heme 3-[18-(2-carboxylatoethyl)-8,13-bis(ethenyl)-

3,7,12, 17-tetramethyl-23H-porphyrin-21-id-2-

yl]propanoate;iron(3?); hydrochloride

53627695 651.9402

Ascorbate (2R)-2-[(1S)-1,2-dihydroxyethyl]-3,4-dihydroxy-

2H-furan-5-one

54670067 176.1241

Cadmium Cadmium 23973 112.4110

Jasmonate 3-Oxo-2-(2-pentenyl)

cyclopentaneacetic acid

5281166 210.2695

Biometals (2014) 27:247–263 249

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[OsAPX–Asc] complex with cadmium (Cd) metal (iv)

[OsAPX–Asc] complex with JA (v) [OsAPX–Asc–JA]

complex with Cd.

PDB viewers

Models were viewed at Molegro Molecular Viewer or

Discovery Studio viewer and useful conclusions

drawn after analysis at Pymol (http://pymol.

sourceforge.net/index.html, DeLano 2002).

Wet-lab experiments

Plant material and growth conditions

Seeds of rice (O. sativa L.) cv. HUR3022 were surface

sterilized with 0.1 % sodium hypochlorite solution and

imbibed in water for 24 h. Seedlings were raised for

10 days in sand cultures, saturated either with Hoagland

nutrient solution (Hoagland and Arnon 1938) that

served as control or nutrient solution supplemented

with either 50 lM Cd(NO3)2 or 5 lM methyl jasmonate

(MeJA) or in combination of 50 lM Cd(NO3)2 and

5 lM MeJA as treatments. Pots were maintained in

growth chamber for 10 day as earlier (Shah et al. 2013).

Seedlings from control and treatments were uprooted at

3, 7 and 10 days intervals, roots and shoots were

separated and used for experiments in triplicate.

Estimation of ascorbate level in root/shoot of rice

Spectrophotometric determination of reduced ascor-

bate levels were carried out at 525 nm according to

Law et al. (1983). Sample cuvettes contained 0.45 ml

plant extract, 0.3 ml trichloroacetic acid (10 %, v/v),

0.3 ml phosphoric acid (44 %, v/v), 0.3 ml bipyridyl

(4 %, w/v) in 70 % ethanol and 0.15 ml ferric chloride

(3 %, w/v). Total ascorbate was measured by reduc-

tion of the oxidized form. The concentration of the

oxidized ascorbate was calculated by the difference

between total and reduced ascorbate and expressed as

lmol g-1 fresh weight.

APX assay

Ascorbate peroxidase (EC1.11.1.11) was assayed as

earlier (Shah et al. 2013). Reaction mixture in a total

volume of 3 ml contained 50 mM potassium phos-

phate buffer (pH 7.0) containing 0.1 mM EDTA and

0.5 mM ascorbic acid, 0.1 mM H2O2 and 0.1 ml

enzyme extract. H2O2 was the last component to be

added and the absorbance was recorded at 290 nm

(extinction coefficient 2.8 mM-1 cm-1). Enzyme

specific activity is expressed as lmol ascorbate

oxidized mg-1 (protein) min-1. Enzyme protein was

determined by the method of Lowry et al. (1951) using

bovine serum albumin (Himedia) as standard.

Results

Sequence analysis of OsAPX

Protein sequence of APX from rice retrieved at NCBI

contained 250 amino acid residues and exhibited 83 %

sequence identity and 90 % similarity with the 261

amino acid long soybean-APX (Fig. 1). The second-

ary structure analysis of OsAPX at PROCHECK

shows it to contain 1 beta hairpin, 2 strands, 14 helices,

30 helix–helix interactions, 16 beta turns and 1 gamma

turn (Fig. 2a).

INTERPRO search revealed that OsAPX contains

IPR10255, IPR002207, IPR002016, IPR019794 and

IPR019793 which represent heme peroxidase and

ascorbate binding domains, explaining its closer struc-

tural resemblance with soybean-APX. Gene Ontology

revealed GO terms GO0006929 responsible for func-

tion during oxidative stress, GO0055114 corresponding

to oxidation–reduction process, GO0004601 for perox-

idase like activity and GO0020037 for heme binding

functions of the OsAPX protein in rice.

Model prediction, optimization and evaluation

Template 1OAF corresponding to soybean-APX

(Fig. 2b) was employed to construct the homology

model for rice APX at Discovery Studio. Of the 25

models obtained the best model for OsAPX (Fig. 2c)

was selected having a dope score -28760.43, verify

score -120.16, expected high score 113.16 and an

expected low score of 50.91. Energy minimization of

the OsAPX model by smart minimizer of DS with

maximum 200 steps resulted in -15975.85 kJ/mol

energy associated with it.

The model of target OsAPX was superimposed on

the structure of the template soybean-APX. An

inspection of the superimposition reveals that overall,

most of the regions of OsAPX are similar to the three-

250 Biometals (2014) 27:247–263

123

Fig. 1 CLUSTALW of cytosolic rice-APX (BAA08264.1)

with cytosolic pea-APX, soybean-APX, cytochrome C-perox-

idase, manganese peroxidase and horse radish peroxidase. 83 %

sequence identity and 90 % similarity between amino acid

sequences of rice and soybean APX is seen. X heme binding site,

substrate binding site, plus Ca2? binding site, asterisks K?

binding site and ash Mn2? binding site

Biometals (2014) 27:247–263 251

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dimensional structure of the template and exhibited an

RMSD score of 0.077 (Fig. 2e). The overall stereo-

chemical quality of the OsAPX enzyme model as

assessed by Ramachandran plot showed 94.2 %

residues in most favoured regions, 5.3 % residues in

additional allowed regions, 0.5 % in generously

allowed regions and 0 % residues in disallowed

regions (Fig. 3a). Energetic architecture of the model

when checked by the proSA energy plot, assesses the

model quality by plotting energies as a function of

amino acid sequence position wherein positive values

reflect problematic area or error in the model. Energy

plot obtained for OsAPX model by ProSA was in

negative area suggesting the model to be of good

quality (Fig. 3b). The OsAPX model when verified

and checked for its quality at ERRAT had a score

89.627 (Fig. 3c) and Verify 3D score of 0.85 implying

good compatibility of the atomic model (3D) of

OsAPX with its amino acid sequence (1D) (Fig. 3d).

The energy minimized OsAPX model thus obtained

was of good quality, reliable and robust which was

submitted with the Protein Model Database online

with PMDBID: PM0078091.

Docking and interaction analysis

Docking of a 3-D model of rice ascorbate peroxidase

with heme cofactor and ascorbate (substrate)

Table 1 lists the accession no., molecular weight and

IUPAC names of the ligands: heme, ascorbate,

cadmium and JA and their corresponding structures

obtained at Molegro are shown as Fig. 4a–d, respec-

tively. These structures were used for docking with the

predicted model of OsAPX. The modeled three

dimensional structure of rice APX docked with heme

porphyrin ?Cd/JA alone or in combination is shown

as Fig. 5a–e.

Fig. 2 a Secondary structure of APX from rice, b 3D model of

soybean APX (1OAF), c 3D model of rice APX obtained by

homology modeling using PDBID: 1OAF as template at

Discovery Studio v3.0 showing a-helices (red) and b-pleated

sheet (blue). The model is submitted with Protein Model

Database (PMDBID: PM0078091), d 3-D structure of rice APX

containing heme and ascorbate (OsAPX holozyme), e Superim-

posed 3-D structures of soybean APX (green) and rice APX

holozyme (red) showing RMSD 0.077. (Color figure online)

252 Biometals (2014) 27:247–263

123

The docking of OsAPX protein backbone with

heme porphyrin is shown as Figs. 5a and 6 OsAPX

possess a heme cavity lined with amino acid residues35Leu, 41Trp, 42His, 65Glu, 132Pro, 134Ala, 163His,167Arg, 169His, 172Arg, 173Ser, 208Asp and 235Tyr. The

heme resides inside this heme binding cavity of

OsAPX by strong H-bond interactions between amino

acid residues 167Arg, 169His and 173Ser present on

OsAPX backbone and heme porphyrin (Fig. 6). The

H-bond interactions occur between OsAPX and heme

involving 167Arg(H)-heme (O4), 169His(H)-

heme(O1), 169His(H)-heme(O2), 173Ser(H)-heme(O1)

and heme(O1)-173Ser(OG) bonds with bond lengths

1.77194, 1.57574, 1.87226, 1.65537 and 2.68242 A,

respectively (Fig. 6). Out of these five H-bonds heme

acts as an e- acceptor in first four bonds and as donor

in the last one. Docking results revealed that H-bond

interactions between OsAPX and heme porphyrin are

critical for proper accommodation of heme inside

OsAPX. In OsAPX the 42His in the distal cavity of

heme group is seen H-bonded with the side chain of71Asn which in turn is hydrogen bonded with the

backbone carbonyl of a glutamic acid (65Glu). At the

closed proximal side of heme, a histidine residue

(163His) is bound to heme iron and forms a strong

hydrogen bond with the buried aspartate residue

(208Asp) (Fig. 6). These six residues observed in this

study were reported to be conserved in all APXs

including pea-APX (Jespersen et al. 1997).

The [OsAPX–Asc] complex is shown as Figs. 5b

and 7. Docking of OsAPX with its substrate ascorbate

resulted in several strong H-bonds between enzyme

OsAPX and ascorbate (Fig. 7a) in the substrate

binding cavity formed by amino acid residues at

position 30KSCAL35, 167RCH169 and 172R (Fig. 1).

Ascorbate gets accommodated in the substrate binding

cavity of OsAPX with the help of three H-bonds near cedge of heme porphyrin involving 30Lys (Fig. 7a). The

bond length for these interactions were 2.129 A

(30Lys(H)-Asc(O1)), 2.37031 A (30Lys(OH)-

Asc(O1)) and 2.03035 A (Asc(H1)-30Lys(O)). Other

interactions with bond lengths between 35Leu(H)-

Asc(O6) (2.39243 A), 167Arg(H)-Asc(O2) (2.26408 A),172Arg(H)-Asc(O4) (1.87913 A), 172Arg-Asc(O5)

Fig. 3 Stereochemical quality of predicted OsAPX model from rice a Ramachandran plot showing 94.2 % residues in favoured region,

b ProSA energy plot in negative area, c ERRAT graph with score 89.625, d Verify 3D graph with score 0.85

Biometals (2014) 27:247–263 253

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(1.94375 A), 169His(NE2)-Asc(H8) (2.39091 A) and

heme(O3)-Asc(O5) (2.86481 A) were also notable

(Fig. 7a). 169His acts as a bridge between heme-

porphyrin and ascorbate and therefore, might help in

the transfer of charge between ascorbate and Fe atom

for redox functions. Histidine is a polar amino acid as

it has a pKa near to that of physiological pH and it is

relatively easy to move protons on and off its side

chain resulting in changes in its side chain from neutral

to positive charge facilitating ideal charge relay

systems.

Docking of OsAPX with cadmium alone

Docking of cadmium with [OsAPX–Asc] complex

resulted in formation of [OsAPX–Asc–Cd] complex

shown as Fig. 5c. The binding of Cd took place at29EKSCAPL35, a site almost similar to the substrate

(ascorbate) binding site 30KSCAPL35 (Fig. 1). As

discussed above ascorbate binds to 30Lys, 167Arg and

169His, but in presence of Cd (i.e. when cadmium

binds to OsAPX) the bond between 169His and

ascorbate breaks (Fig. 7) and two new bonds are

formed instead, between heme(O3) and ascorbate(O5)

and heme (O3) and ascorbate(H8) (Fig. 7b). This

suggests that cadmium alters an important contact

between heme and ascorbate present on OsAPX,

shifting thereby the protein conformation and disturb-

ing the redox reaction. In the [OsAPX–Asc] complex

the ascorbate forms a hydrogen bond between O5 of

ascorbate (acceptor) and O3 atom of heme group

(donor) with bond length of 2.86481 A and angles

141.304 (AHY) and 114.651(DHA) (Fig. 7a). In

presence of cadmium however, the ascorbate forms

two H-bonds with the heme group, one between O3

and O5 with bond length 2.86481 A and angles

141.304 and 22.9921 in which heme acts as donor and

another between H8 of ascorbate and O3 of heme with

bond length of 1.95796 A and angles 145.144 and

135.426 wherein ascorbate acts as donor (Fig. 7b).

Fig. 4 Chemical structures

of a Heme porphyrin,

b Ascorbate, c Cadmium,

d Jasmonate obtained at

Discovery Studio

254 Biometals (2014) 27:247–263

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The bonds formed between OsAPX-29Glu and Cd

were longer in bond lengths than those reported for

OsAPX-30Lys and ascorbate (Fig. 7a). Cadmium

exhibited four electrostatic interactions involving

CA, C, O and H groups of 29Glu(E) amino acid

residue on OsAPX backbone with bond lengths of

4.9952, 4.7272, 3.8463 and 4.54298 A respectively

within the ascorbate binding cavity (Fig. 8).

Docking of OsAPX with jasmonate

The [OsAPX–Asc–JA] complex is seen as Fig. 5e.

Binding of [OsAPX–Asc] complex to JA revealed

binding positions 38Arg, 41Trp, 42His, 68His, 72Ala,128Glu, 133Asp, 136Lys, 140His, 163His, 172Arg, 173Ser

which are residues, forming the polar hydrophilic

cavity. Jasmonate binds to OsAPX through H-bonds

formed between 38Arg (H) and JA(O1) with bond

length 1.73012A and 134Ala(H) and JA(O3) atom of

JA with bond length 2.07035 A (Fig. 9). Results

suggest no significant changes in [OsAPX–Asc]

complex in presence of JA alone in rice.

Docking of [OsAPX–Asc–JA] complex with cadmium

Jasmonate enhanced the activity of OsAPX and lowered

the effect of Cd under wet-lab conditions (communi-

cated elsewhere). It is therefore expected that Cd binds

at a position other than that of the substrate binding site

when JA is present in the medium so that the normal

biological function of the OsAPX-enzyme is not

inhibited upon exposure to Cd. It is quite possible that

JA competes for the same position as that of Cd and does

not allow Cd to bind. To verify whether JA actually

plays a direct role in restoring the activity of OsAPX, in

presence of Cd the binding of [OsAPX–Asc–JA] com-

plex with Cd were performed. [OsAPX–Asc–JA–Cd]

complex obtained at Molegro is shown as Fig. 5e.

In presence of JA, the heavy metal cadmium binds

at the surface of OsAPX-protein, the binding position

Fig. 5 a Native APX enzyme, b Native APX enzyme-substrate

complex, c Native APX enzyme-substrate complex bound to

cadmium, d Native APX enzyme-substrate complex bound to

jasmonate, e Native APX enzyme-substrate complex bound to

cadmium in presence of jasmonate

Biometals (2014) 27:247–263 255

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being different and very distant from ascorbate

binding site. In the OsAPX–Asc–JA–Cd] complex

Cd gets accommodated in a loop region at the surface

of OsAPX protein through electrostatic interactions

involving 3Lys, 5Tyr, 240Leu, 241Lys and 244Glu

residues (Fig. 10).

Level of ascorbate and activity of rice ascorbate

peroxidase (OsAPX) in presence of Cd and/or JA

in rice tissues under wet-lab experiments

Table 2 shows the level of ascorbate and activity of

APX in roots/shoots of rice exposed to Cd in presence

or absence of JA at increasing days of growth. It is

evident that the total ascorbate (AsA) levels remained

nearly constant in both roots/shoots of control plants

either in presence or absence of JA. 50 lM Cd2?

alone led to *1–1.2 fold decrease in ascorbate levels

in roots/shoots of rice as compared to controls.

Application of 5 lM JA ? Cd2? improved the ascor-

bate content in roots as well as shoots of Cd-stressed

rice throughout the growth period (Table 2).

Cadmium exposure alone decreased the activity of

OsAPX by 22–27 % in shoots and 15–38 % in roots at

3, 7 and 10th day of growth in growing rice seedlings.

Application of exogenous JA to Cd-stressed rice

increased OsAPX activity in shoots as well as roots by

*12–21 % at increasing days of growth (Table 2)

suggesting a mitigating effect of JA on Cd-toxicity in

rice.

Discussion

Many metalloenzymes operate by storing oxidizing

equivalents derived from O2 or peroxides in the active

site to form relatively stable organic radicals and/or

high-valence metal complexes. A high level of

endogenous ascorbate is essential for effectively

maintaining the antioxidant system that protects plants

from oxidative damage due to biotic and abiotic

stresses (Shigeoka et al. 2002). APX activity and

ascorbate content show identical reductions in pea

plants under water-deficit or paraquat induced stress

(Escuredo et al. 1998).

Recent studies have focused on the changes in

activities of APX isoenzymes in higher plants sub-

jected to several environmental stresses such as ozone,

Fig. 6 The OsAPX-heme

complex (red) showing

H-bond interactions

between amino acid residues

of APX (blue) and heme

porphyrin (red), involving167Arg, 169His and 173Ser.

Sphere represents heme-Fe.

(Color figure online)

256 Biometals (2014) 27:247–263

123

high light, and extremes of temperatures, salt and

paraquat (Tanaka et al. 1985; Mittler and Zilinskas

1991; Prasad et al. 1994; Rao et al. 1996; Lopez et al.

1996; Yoshimura et al. 2000; Nahakpam and Shah

2011). APX activities generally increases along with

activities of other antioxidant enzymes like catalase,

SOD and GSH reductase in response to environmental

stresses suggesting that the components of ROS-

scavenging systems are co-regulated (Shigeoka et al.

2002; Shah et al. 2013). As structural information has

appeared it is becoming clearer as to what strategies is

employed by OsAPX to adapt their structural frame-

works to bind ascorbate or metal in presence or

absence of JAs in rice plants grown under Cd-stress.

Heme peroxidases occupy a unique interface

between biology and chemistry that continues to

fascinate scientists as much now as it did decades ago.

Plant peroxidases contain two ion-binding sites one

proximal and one distal to the heme plane which

usually binds calcium thereby stabilizing heme envi-

ronment. The crystal structure of cytosolic-pea APX

however, possesses a single cation-binding site,

believed to be occupied by potassium in the distal

domain (Patterson and Poulos 1995). This distal/

proximal calcium is reported to be important for

maintenance of heme environment and the enzyme

stability. Similar to the results of this study the APX

isoenzymes in higher plants are reported to show high

homology (70–90 %). The 38Arg, 71Asn, 65Glu and208Asp residues around the distal 42His and proximal163/169His are highly conserved throughout the plant

peroxidase family (Shigeoka et al. 2002). These

residues are essential for binding of the ligand heme

(Welinder 1992). In addition 41Trp and 179Trp are

conserved in most APX and participate in hydrogen

bonding network together with 163His and 208Asp

residues (Jespersen et al. 1997).

The ascorbate binding site close on APX which is to32Cys and c-edge is a high affinity site whereas at the

d-meso position is the low-affinity site (Hill et al.

1997). The ascorbate oxidation however, is reported

not to occur at the exposed heme edge but at an

Fig. 7 a [OsAPX-Asc] complex showing the bonds formed and

their bond lengths between (i) heme(O3) and Asc(O5)

(2.86481A) (ii) OsAPX-169His(NH1) and heme(O1) (iii)

OsAPX-169His(ND1) and heme(O2) (iv) OsAPX-169His(NE2)

and Asc(H8), b [OsAPX-Asc-Cd] complex showing two new

bonds formed between heme(O3) with Asc(O5) (2.86481A)

(v) and (H8) (1.95796A) (vi). The absence of OsAPX-169His is

clearly visible. Cd metal is represented as blue sphere and

Heme-Fe is represented as red sphere

Biometals (2014) 27:247–263 257

123

alternate binding site in the vicinity of 32Cys and the

propionates (Mandelman et al. 1998). The proximal

metal binding site of OsAPX (164Thr, 180Thr and187Asp), is similar to that reported for pea APX and is

invariant probably in all types of APX. The potential

distal metal binding site in the distal domain (position

43, 57 and 59 of pea APX) is occupied by metal ion in

all APXs (Welinder 1992). Substrate binding in APX

has emerged to occur at two kinetically competent but

separate locations (Lad et al. 2002; Sharp et al. 2004;

Gumiero et al. 2010). The first, close to the 6-propi-

onate c-heme edge close to 32Cys and 172Arg, the

(C15) position of the heme (Fig. 4a) and other at

d-heme edge together with hydrogen bonding inter-

actions involving Arg/Lys/His residues and the heme

propionates. In a 1:1 complex formed between ascor-

bate and APX with modified phenyl hydrazine heme

group, the ascorbate bound close to c-meso involving134Ala residue as also observed in OsAPX-ascorbate

complex, herein.

The crystal structure of APX including active site

architecture is very similar to cytochrome-C-peroxi-

dase (Bonagura et al. 1996) and to pea-APX (Jesper-

sen et al. 1997). A cation located *8 A from the

proximal Trp is present in APX, but is absent in cyt-C-

peroxidase. Electrostatic factors and the presence of

metal ions affect the relative stability of the Trp in

cytochrome c peroxidase (Patterson and Poulos 1995).

A study of substrate binding in APX suggests that the

binding site remains competent for binding of

Fig. 8 The magnified view

of [OsAPX-Asc-Cd]

complex showing binding of

Cd metal via 29Glu and30Lys of OsAPX. The four

electrostatic interactions of29Glu (CA, C, O and H) with

Cd is observed in the

complex which leads to

cleavage of 169His(NE2)-

Asc(H8) bond shown in

Figs 5 and 7. The bond

lengths of each bond

interaction is indicated in A

258 Biometals (2014) 27:247–263

123

ascorbate even in absence of both 172Arg and 30Lys

(MacDonald et al. 2006). Similar to the crystal

structure of APX/ascorbate complex (1OAF) from

soybean (Sharp et al. 2003) the [OsAPX–Asc] com-

plex also bound ascorbate at the c-heme edge through

hydrogen bonds to 30Lys, 172Arg and the heme 6

propionate. Functional amino acid residues which are

related to the binding of heme and ascorbate have been

identified by mutation analysis in recombinant pea.172Arg of pea cytosolic APX has an important role in

ascorbate utilization (Bursey and Poulos 2000). 38Arg

has a functional role in the control of substrate binding

and orientation (Celik et al. 2001). This suggests that

the substrate oxidation probably occurs by the electron

delivery through the heme propionates in OsAPX.

It is also known that 30Lys is not conserved across

all APXs (Jespersen et al. 1997). Where 30Lys is not

present, it is replaced by a Lys residue at position 31 or

29. Hence 30Lys (or equivalent residue at position 31

or 29) may not be critical for ascorbate oxidation in all

cases (MacDonald et al. 2006), which is also consis-

tent with our observation. No 30 Lys residue is present

in spinach-APX but 172Arg is present and data show

that H-bonds between the bound ascorbate and

enzyme involving 172Arg more influential and have

major role in controlling substrate binding (MacDon-

ald et al. 2006).

Heme exhibits great sensitivity towards its local

environment. A pH dependent replacement of proxi-

mal histidine ligand by smaller amino acids that cannot

coordinate heme iron is known (Smith and Veitch

1998). These workers suggested that the proximal

aspartate residue modulates the ability of APX-enzyme

to bind ligands at the vacant 6th coordination site of the

heme group (Veitch and Smith 2001). Smulevich and

co-workers (1989) through site directed mutagenesis

studies have shown that on the proximal side, the175His-235Asp H-bond is a critical interaction, modu-

lating the Fe–His bond strength and restraining the Fe

atom from moving into the heme plane and binding a

distal water molecule. These researchers suggested the

interactions to be of double-well potential with nearly

isoenergetic minima, wherein the coordinate involving

proton transfer is coupled to the conformation.

In [OsAPX–Asc–JA] complex, it is seen that Cd

binds at a position very different to its predicted

Fig. 9 a [OsAPX-Asc-JA] complex formed between [OsAPX-Asc] and JA, b H-bond interactions between (i) OsAPX41Trp(H)-

JA(O2) (ii) heme(N1)-JA(O2) (iii) heme(N1)-JA(H11) (iv) heme(N2)-JA(O2) and heme(N2)-JA(H1)

Biometals (2014) 27:247–263 259

123

binding sites (29E-35L). At the same time when JA is

present along with Cd it restores the bonds formed

between OsAPX–Asc as well as Asc-heme. It is also

noted that Cd is unable to replace JA in the complex

and resides only at the surface through weak electro-

static interactions in a cavity rich in negatively

charged amino acids namely Asp and Glu at the core.

Protein–protein interfaces or regions near interfaces

seem to be inherently flexible or intrinsically disor-

dered allowing Cd to penetrate and displace the

protein interaction partner. Additionally it is also

reflected that Cd do not cover the entire protein–

protein binding interface but rather target only a small

number of interface residues, which contribute the

most to the altered binding energy obtained. Manga-

nese–peroxidase binding site is hexa-coordinate with

four carboxylate ligands from the side chain of three

amino acids 179Asp, 35Glu and 39Glu and heme

propionate and Cd has been reported to stabilize Mn-

POX in P. chrysoporium. Infact Cd metal was shown

to compete for the same site as that of Mn2? (Youngs

et al. 2000). Cd was bound at the C-terminus in the Cd-

manganese peroxidase crystal formed by only two

amino acid ligands to the metal suggesting a second

Fig. 10 The [OsAPX-Asc-JA-Cd] complex showing that in

presence of JA, Cd binds on the surface of APX and the H-bonds

are formed between (i) OsAPX38Arg-JA(O1) (ii) OsAPX134

Ala(H)-JA(O3) and (iii) heme(O3) and Asc(O5). Blue (large)

and red (small) spheres represent Cd and heme-Fe, respectively.

(Color figure online)

260 Biometals (2014) 27:247–263

123

low affinity metal-binding site on Mn-POX (Youngs

et al. 2000). Spectroscopic studies also indicated

alternative or additional sites for Cd binding on

manganese peroxidase. There is also evidence that

Cd can substitute Ca in peanut peroxidase (Patterson

and Poulos 1995).

The present data are useful in terms of our wider

understanding of ascorbate binding in OsAPX. The

peptide segment residues 69–72, 131–135 and

171–175 from the surface of the active site entrance

channel near the heme edge of OsAPX are perhaps

essential to the interaction of APX with ascorbate

(Fig. 1). These segments are characterized by XANX,

LPDAX and (E) RSGF/W respectively in OsAPX. The

catalytic domain of APX is conserved in all members

of the plant peroxidase superfamily and consists of

241–273 residues folded into ten or more character-

istic alpha-helices reported initially in pea-APX

crystal structure (Patterson and Poulos 1995). Seven

charged residues, which participate in electrostatic

interactions in the dimer interface of pea cytosol-APX

are highly conserved in all soluble cytosol-APX

including rice except for 21Gln and 112Ala in OsAPX

and 10Thr in cotton-APX (Patterson and Poulos 1995).

Presence of 43Asp, 57Asn and 59Ser as in OsAPX are

characteristic of all APXs except soluble cytosol cs/

and cs2, whereas fungal and plant peroxidases have43Asp, 57Asp and 59Ser instead (Jespersen et al. 1997).

Results clearly indicate that OsAPX has 250 amino

acids and in the presence of JA the restoration of the

[OsAPX–Asc] binding occurs which is otherwise

altered in presence of cadmium metal alone. Although

care has been taken in performing the docking studies

nevertheless for more understanding of enzyme

behavior under various abiotic stresses MD simula-

tions would be helpful. It is the interaction between

heme and ascorbate which is modulated differently in

presence of Cd/JA. In absence of JA, Cd binds to the

[OsAPX–Asc] complex at the proximal end of APX

near Asc-binding site, whereas in presence of JA, it

binds on the opposite side of the Asc-binding site

involving 29Glu and 30Lys residues. Our in silico

binding studies well correlate with the wet lab results

where exogenous application of JA increased the

activity of OsAPX in rice grown under Cd stress and

therefore, it can be concluded that the activity of

OsAPX in rice roots and shoots is compromised under

Cd-stress alone, however addition of JA restores and

maintains the stability of OsAPX in rice plants.Ta

ble

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7

Biometals (2014) 27:247–263 261

123

Acknowledgements Authors are grateful to Banaras Hindu

University for providing infrastructural facilities for

computational studies. IS is thankful to DST, Govt. of India

for DST-Women Scientist Fellowship.

Conflict of interest There is no conflict of interest.

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