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Redox Metalloproteins. These are proteins in which the bound metal ions undergo changes in their oxidation states that are an essential part of its biological function. The questions that we will address are: which metal ions are involved? what is the structure of the metal-cofactor?

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redox metalloproteins
Redox Metalloproteins

These are proteins in which the bound metal ions undergo changes in their oxidation states that are an essential part of its biological function

  • The questions that we will address are:
  • which metal ions are involved?
  • what is the structure of the metal-cofactor?
  • how does the protein incorporate and utilize these cofactors?
redox potentials
Redox potentials

Selected redox active metal ions

The higher the Eo values the more energy is required for reduction

Many of the enzyme-catalyzed redox reactions involve oxygen

Oxygen species

comparison of iron and copper redox proteins
Comparison of iron and copper redox proteins

Both Cu and Fe proteins are capable of supporting a wide range of similar biological functions

heme peroxidases and catalase
Heme peroxidases and catalase

Each of these enzymes use an Fe-heme redox cofactor

How do proteins fine-tune an Fe-heme cofactor to support different redox reactions?

By changing the identity of the axial ligand(s)

catalase
Catalase

This is a protective enzyme that disproportionates highly reactive peroxide into less reactive oxygen species

Peroxide binds in the axial position of the Fe-heme cofactor

cytochromes
cytochromes

Functions in electron transfer reactions that shuttle electrons to successive electron carriers

Unlike the situation with catalase there is no need for a substrate binding site, so both axial positions are occupied by protein ligands

cytochrome c
Cytochrome c

An electron donor protein will dock with cytochrome c and transfer an electron to Fe(III), thereby reducing it to Fe(II)

Binding of an artificial probe allows mapping of the electron transfer pathway

cytochrome c oxidase
Cytochrome c Oxidase

Cytochrome oxidase is the terminal electron acceptor in the electron transport pathway

It catalyzes the reduction of O2 to H2O, which is a net four electron change

To do this requires sufficient redox capacity to accept and then to transfer a total of four electrons

Cytochrome oxidase contains two Fe-heme and two Cu sites, each of which can accept and donate a single electron

cytochrome c oxidase10
Cytochrome c Oxidase

Cytochrome oxidase is a membrane-spanning protein

Oxygen binds from one side of the membrane and water is released to the other side

There are additional metal ion binding sites on the surface when the enzyme is crystallized with either Cd or Zn salts

These sites map the negative surface where cytochrome c binds to transfer electrons

The redox core of the enzyme consists of the Fe-heme and Cu centers

Proc. Natl. Acad. Sci.104, 7881 (2007)

functions of iron sulfur centers
Functions of Iron-Sulfur Centers
  • photosynthesis
  • cell respiration
  • nitrogen fixation
  • metabolism of hydrogen
  • NO2 and SO3 oxidation & reduction
  • redox catalysis
iron sulfur clusters structural types
Iron-sulfur clustersstructural types

The simplest Fe/S cluster consists of a single Fe coordinated to four cysteines

More complex Fe/S clusters have inorganic sulfur atoms bridging between adjacent Fe atoms

2Fe/2S and 4Fe/4S clusters are the most common

The 3Fe/4S clusters are frequently activated by addition of another Fe

iron sulfur clusters representative examples
Iron-sulfur clustersrepresentative examples

The charges represent the most common oxidation states for each cluster

iron sulfur proteins spectroscopic properties
Iron-sulfur proteinsspectroscopic properties

Changes in the environment around each type of cluster are used to fine-tune the redox potentials

rubredoxin 1fe 0s cluster
Rubredoxin1Fe-0S cluster

High-spin, ferric iron by EPR and Mossbauer spectroscopy

Redox potential of rubredoxins range from -50 to +50 mV

ferredoxin 2fe 2s cluster
Ferredoxin2Fe-2S cluster

High-spin, ferric iron magnetically coupled (EPR silent)

4fe 4s cluster
4Fe-4S Cluster

distorted cubic shape

bound to the protein through

four cysteine thiolates

hydrogenase
Hydrogenase

H2 ―> H- + H+

―> 2 H+ + 2 e-

This enzyme catalyzes the disproportionation of unreactive H2 gas to hydride ion and a proton

A subsequent two electron oxidation of hydride to a proton completes the reaction

This enzyme contains multiple Fe-S clusters that act as an electron shuttle pathway

In addition, there is a non-heme Fe, Ni and Mg bound in close proximity

hydrogenase active site
Hydrogenaseactive site

The enzyme contains bound Fe (red) and Ni (green) in a binuclear center

Hydrogen coordinates to the axial position on Fe

This structure shows CO bound in place of H2 and a bridging hydroxide between Fe and Ni

hydrogenase other forms
Hydrogenaseother forms

In addition to this Ni/Fe form, two other types of hydrogenases have been characterized from other species

All three forms contain an active site Cys, bound CO and either CN or pyridinol

The [NiFe] and [FeFe] forms use Fe-S clusters as redox partners, while the [Fe] form uses a methanopterin

Science 321, 572 (2008)

ribonucleotide reductase
Ribonucleotide reductase

conversion of ribose to deoxyribose

Important for the production of deoxynucleotides that serve as the building blocks for DNA synthesis

This enzyme uses a binuclear iron center as well as a tyrosyl radical to catalyze the removal of the 2’-hydroxyl group

ribonucleotide reductase25
Ribonucleotide reductase

Bimetallic center with bridging glutamate and oxygen ligands

ribonucleotide reductase altered metal ion binding
Ribonucleotidereductasealtered metal ion binding

binding of diiron in RNR

binding of dimanganese in RNR

This alternative form of RNR can be used by pathogenic organisms when iron supplies are limited, such as during human infection

The iron form of RNR can be oxidized by O2 to initial the radical reaction, but the manganese form cannot be oxidized by oxygen

This shortcoming requires production of an additional protein to activate the dimanganese version of RNR

Biochemistry49, 1297 (2010)

ribonucleotide reductase activation of the mn 2 form
Ribonucleotidereductaseactivation of the Mn2 form

This accessory protein (NrdI) generates a flavin peroxide species (yellow structure) by reaction with O2

After NrdI docks with RNR the peroxide is proposed to travel through a cavity in the enzyme (blue mesh) to oxidize the Mn ions and activate the enzyme

Science329, 1526 (2010)

hydroxylase
Hydroxylase

The oxidation of unreactive methane is a difficult process

The enzyme uses a binuclear Fe center to active the hydroxide nucleophile and to bind the reactant

2-bromoethanol

3-chloropropanol

The alcohol hydroxyl group is bound across both Fe atoms

The Cl at the end of longer substrates can be bound in several orientations

copper centers in proteins
Copper Centers in Proteins

coordination geometry

structure and function

plastocyanin
Plastocyanin

Functions as an electron carrier in photosynthesis

Plastocyanin is a low molecular weight protein that shuttles electrons between membrane-bound photosystems I and II (analogous to cytochrome c in mammalian electron transport)

This is an early structure of a plant plastocyanin showing the presence of a Cu binding site

plastocyanin32
Plastocyanin

Cu is coordinated to two His, a Cys and a Met

There are only slight changes in the bond distances (and coordination geometry) as Cu cycles between the +1 and +2 oxidation states

The coordination geometry and bond distances are a compromise between the preferences for Cu in the different oxidation states

azurin
Azurin

Azurin is a blue-copper protein (type I copper)

Cu is coordinated in a distorted trigonal bypyramid

Two His (H46 & H114), a Cys (C112), a Met (M121) and a carbonyl group provide donor atoms

The intense blue color comes from a charge transfer band arising from the short Cu-S bond to Cys

azurin redox tuning
Azurinredox tuning

native azurin structure

N47S/F114N mutant

alters H-bond to C112 and introduces H-bond to N114

F114P/M121Q mutant

deletes H-bond to C112

Nature462, 113 (2009)

azurin redox tuning35
Azurinredox tuning
  • Changes in hydrogen bonding patterns can lead to dramatic changes in redox potentials
  • Altering the hydrophobicity of the axial Cu ligand (M121) leads to a linear increase in E0 values
  • Replacing F114 with P removes a H-bond to C112 and lowers the E0 value (black line)
  • Introducing S47 and N114 increases the H-bonding to C112, stabilizing Cu(I) and increasing the redox potential (blue and red lines).

Nature462, 113 (2009)

superoxide dismutase
Superoxide Dismutase

Catalyzes the disproportionation of highly reactive superoxide radical into peroxide and oxygen

Catalase then deals with the peroxide that is produced

Superoxide Dismutase comes in two different forms, both of which are metalloenzymes

The Mn form of SOD cycles the metal between the +2 and +3 oxidation states

The Cu-Zn form of SOD cycles the Cu between the +1 and +2 oxidation states

superoxide dismutase cu zn form
Superoxide DismutaseCu-Zn form

Cu and Zn are bound in adjacent sites and share a common histidine

The oxygen reactants bind at the Cu site for the redox reaction

superoxide dismutase38
Superoxide dismutase

The Cu-Zn enzyme is a dimer with identical active sites in each subunit

The Zn site is 4-coordinate, tetrahedral with 3 His & 1 Asp

The Cu site is also 4-coordinate with 4 His

This site expands to 5-coordinate square pyramidal when the substrate binds

multielectron pair redox reactions
Multielectron pairredox reactions

The previous redox reactions each involve the transfer of either a single electron or a pair of electrons

Some redox reactions require the transfer of more than one electron pair

Respiration

Cytochrome oxidase reduction of oxygen to water

Photosynthesis

oxidative conversion of water to oxygen

Catechol oxidation

insertion of oxygen into aromatic compounds

These are each redox reactions involving a net change of four electrons

photosynthetic reaction center
Photosynthetic Reaction Center

There are multiple electron carriers that will funnel electrons into the active site

catechuate dioxygenase
Catechuate dioxygenase

Unlike the photosynthetic reaction center this enzyme uses a single Fe site for a net four electron reaction

This will clearly require multiple electron transfer cycles that go through some partially oxidized intermediates

multielectron pair redox reactions43
Multielectron pairredox reactions

There are even more challenging redox reactions that involve net six electron changes

Nitrogen fixation

conversion of inert nitrogen gas into ammonia for incorporation into metabolism

Sulfite reduction

production of hydrogen sulfide for use as a reductant in anaerobic organisms

Nitrite reduction

production of ammonia

Each of these transformations requires sufficient redox capacity for a 6-electron reduction

molybdenum containing redox enzymes
Molybdenum containing redox enzymes

Because Mo has a wider range of stable oxidation states than either Cu or Fe it is frequently employed in multielectron redox reactions

nitrogenase structure
Nitrogenasestructure

This enzyme uses two different types of subunits with different metal cofactors to catalyze nitrogen fixation

Mo-Fe protein

Fe protein

nitrogenase mo fe cofactor
NitrogenaseMo-Fe cofactor

The Fe protein contains a traditional 4Fe/4S cluster

The Mo-Fe protein has a unique cofactor

This cofactor is constructed from two 4Fe/4S clusters

These clusters are linked together by three bridging sulfurs

In addition, one of the Fe is replace by a Mo

sulfite reductase
Sulfite reductase

How are 6 electrons accommodated in the enzyme ?

The enzyme has two separate metal cofactors

sulfite reductase48
Sulfite reductase

Phosphate, acting as a sulfite mimic, binds to the Fe-heme displacing the water

The Fe/S cluster is located on the opposite face of the heme ring

There are multiple positively-charged amino acids that aid in sulfite binding

Electrons are channeled through the 4Fe/4S cluster to the sulfite bound at the Fe-heme

Handbook of Metalloproteins

Wiley & Sons, pp. 471-485 (2001)

metalloprotein ligands
Metalloprotein Ligands

We have seen the use of a wide variety of metal ions carrying out many different tasks in metalloproteins

A summary of known metal ion binding sites shows the most common coordination numbers and ligand types for each of the frequently seen metal ions

summary
Summary
  • Redox metalloproteins use metal ions that can undergo reversible oxidation/reduction
  • Both iron and copper can undergo one electron redox reactions
  • Iron redox proteins include heme, Fe/S, and non-heme iron centers
  • Copper redox proteins fall into three classes with different electronic properties
  • Cytochromes are involved in one electron transfer processes
  • Ribonucleotide reductase uses both a binuclear iron center and a tyrosyl radical
  • Multiple electron pair redox reactions require additional redox centers (nitrogenase) or successive electron transfers (sulfite reductase)