Oxidative stress Vadim Gladyshev Redox Biology Center, University of Nebraska.

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Oxidative stressVadim Gladyshev

Redox Biology Center, University of Nebraska

Origin of oxidative stress

Formation of earth: ~4.5 billion years

Chemical evidence of life on earth : ~3.85 billion years ago

Initially, the Earth had a reducing environment

- Gaseous mixtures of NH3, CH4, H2O, H2

- No molecular oxygen, excess metals

Oxygen is a potent oxidant- Easy transfer of electrons to oxygen - Oxidative metabolism (respiration)

Oxygen toxicity and reduced solubility of metals

Redox Biology and the Evolution of Life

Utilization of Oxygen by Organisms

All animals and plants (and ancestral eukaryote) use oxygen to

generate energy

2~3 billion years ago, probably due to the evolution of oxygen-

evolving photosynthetic organisms

A prevalent element (53.8% atomic abundance in the earth’s crust,

21% in atmosphere)

Oxygen is soluble in pure water (surface water is generally in

equilibrium with the atmosphere)

However, diffusion of oxygen through tissues is very low (evolution of

oxygen transfer mechanism)

Intermediates of oxygen metabolism are also utilized for physiological

purposes (e.g. signaling).

Oxidative stress

High Low

Oxygen Toxicity

Generation of reactive oxygen species

A free radical is any species capable of independent existence

that contains one or more unpaired electrons

Sources of oxygen free radicals (reactive oxygen species)

- Mitochondrial electron transport chain

- Transition metal-mediated reactions

- Designated systems for ROS generation

Reactive oxygen species-mediated reactions

Moderately or highly reactive

Fe2+ (Cu+) + H2O2 <-> HO. + HO- + Fe3+ (Cu2+)

Neutrophil-mediated killing of bacteria

Adaptations to Oxygen Toxicity

Anaerobic life

Defense mechanisms against oxygen toxicity

Prevention of generation of reactive oxygen species

- Metal sequestration

Antioxidants and antioxidant enzymes

- Scavenge reactive oxygen species

Damage repair systems

- DNA and protein damage repair

Cu Zn Superoxide Dismutase

Catalase

Peroxiredoxin

Signaling by hydrogen peroxide

Oxidation of Cys residues as the basis for peroxide signaling

Nitric Oxide

CO is an important regulator of hypoxic sensing by the carotid body

Are antioxidants effective in human health and disease?

Among proteins with functional Cys, some utilize this residue for redox catalysis:

thiol oxidoreductases

MsrA MsrB fRMsr

TRX GRX

variety of unrelated

Thiol oxidoreductases

The main fold:Thioredoxin fold

(3 layers, a/b/a; mixed beta-sheet of 4 strands, order 4312)

Thiol oxidoreductases - catalytic and resolving Cys residues

PDI MsrB1 fRMsr 1-Cys Prx

Two types of redox active Cys: Catalytic Cys (k) and Resolving Cys (r)

Different organization of resolving Cys in thiol oxidoreductases

Selenocysteine (Sec) in proteins: Sec is always placed in the active site, and it serves the function of the catalytic redox Cys

Fomenko et al (2007)

Thiol oxidoreductases: catalytic Cys and Sec

Involved in many biochemical processes and play central roles in redox homeostasis

Thiol oxidoreductases - functions

Thioredoxin system: TRXs, TR..

Glutathione/Glutaredoxin system: GRXs, GR..

Removal of ROS: AhpC, PRXs..

Met oxidative stress repair: MsrA, MsrB ..

Formation of disulfide bonds: Dsbs, Ero1, PDI …

V ≈ -250 mV

V ≈ -150 mV

Structure / AA composition around catalytic Cys: Modulation of pKa and Redox potential

Normal

Apoptotic

Cys modificationsTrans-nitrosylation: effects on apoptotic pathways

GSNO-protein AND protein-protein interaction Specificity

Trace elements (micronutrients)

Precursor Z

Molybdopterin

Mo-MPT

(Moco)

Nitrogenase (Fe-Mo)

ModABC

WtpABC

TupABC

Molybdenum (Mo)

- Prokaryotes

- Eukaryotes

Sulfite oxidase (SO)

Xanthine oxidase (XO)

Dimethylsulfoxide reductase (DMSOR)

Aldehyde:ferredoxin oxidoreductase (AOR)

Chicken sulfite oxidase

Sulfite oxidase active site

3D structure

(PDB code: 1SOX)

Copper (Cu)

- Prokaryotes

Cytochrome c oxidase subunit I (COX I)Cytochrome c oxidase subunit II (COX II)Plastocyanin family

Azurin familyRusticyanin (RC)Nitrosocyanin (NC)Nitrous oxide reductase (N2OR)NADH dehydrogenase 2 (NDH-2)

[Cu,Zn] superoxide dismutase (CuZn SodC)Copper amine oxidase (CuAO)

Particulate Methane monooxygenase (pMMO)

Multicopper oxidases (MCOs)

NiR, CueO, laccase, bilirubin oxidase, etc.

Tyrosinase

CutCCutF

CusCBA

CtaA (Cyanobacteria)

[Cu,Zn] superoxide dismutase (CuZn SodC)Copper amine oxidase (CuAO)Multicopper oxidases (MCOs) Laccase, Fet3p, hephaestin, ceruloplasmin, etc.

Plantacyanin (PNC)

Umecyanin, mavicyanin, stellacyanin, etc.

Peptidylglycine alpha-hydroxylating monooxygenase (PHM)Dopamine beta-monooxygenase (DBM)HemocyaninCnx1GGalactose oxidase (GAO)

- Eukaryotes

Cytochrome c oxidase subunit I (COX I)Cytochrome c oxidase subunit II (COX II)Plastocyanin family

Tyrosinase

Copper (Cu)

ATPADP

CusCBA

Cu(II)

CueOCu(I)

Cu(II)

Cu(I) or Cu(II)

Blue copper proteins

Cu homeostasis in bacteria

Cu homeostasis in eukaryotes

CCS chaperone

Cu-Zn SOD

Mitochondrion

Cu ion

Nucleus

Tyrosinase

Human Cu-Zn SOD

copper (blue-green sphere) and zinc (grey spheres)(PDB code: 1HL5)

Nickel (Ni) and cobalt (Co)

Urease

Ni-Fe hydrogenase

Carbon monoxide dehydrogenase (Ni-CODH)

Acetyl-coenzyme A decarbonylase (CODH/ACS)

Ni-containing superoxide dismutase (SodN)

Methyl-coenzyme M reductase (MCR)

Nik/CbiM

Nik/CbiN

Nik/CbiQ

Nik/CbiMNQO (Nik/CbiKMLQO)

Nik/CbiL

Nik/CbiK

Nik/CbiM

Nik/CbiQ

A NikC Nik

NikABCDE

HupE/UreJ

Vitamin B12

(cobalamin)

B12-dependent isomerase - Methylmalonyl-CoA mutase (MCM)

- Isobutyryl-CoA mutase (ICM)

- Glutamate mutase (GM)

- Methyleneglutarate mutase (MGM)

- D-lysine 5,6-aminomutase (5,6-LAM)

- B12-dependent ribonucleotide reductase II

- Diol/glycerol dehydratase (DDH/GDH)

- Ethanolamine ammonia lyase (EAL)

B12-dependent methyltransferase - B12-dependent methionine synthase (MetH)

- B12-dependent methyltransferases

Mta, Mtm, Mtb, Mtt, Mts, Mtv and Mtr

B12-dependent dehalogenase

Overview of trace Overview of trace element utilizationelement utilization

• Cu utilization is widespread in bacteria and eukaryotes, but restricted in archaea

• Only a few organisms utilize all five trace elementsBacteria: 94Archaea: 3Eukaryotes: 9

• >50% prokaryotic organisms use the four metals

• Only 9 eukaryotes use the four metals

• Many Saccharomycotina lost the ability to use most of the five trace elements

Cu Ni Co (B12) Mo Se (Sec)Phyla

Total 432(80%) 319(59%) 410(76%) 401(74%) 139(26%)

Total 26(55%) 39(83%) 45(96%) 46(98%) 6(13%)

Total 154(96%) 51(32%) 49(31%) 105(66%) 76(48%)

Bacteria

Archaea

Eukarya

O. sativa (76)

O. sativa (13)

D. rerio (34)

D. discoideum (3)

Fungi MetazoaViridiplantae

• Land plants possess the largest Mo- and Cu-dependent metalloproteomes in eukaryotes

Metalloproteomes and selenoproteomes in eukaryotesMetalloproteomes and selenoproteomes in eukaryotes

Co(B12)

Se(Sec)

Are antioxidants effective in human health and disease?

Oxidative stress Vadim Gladyshev Redox Biology Center, University of Nebraska.

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