Redox Metalloproteins

1. 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?

2. 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

3. Comparison of iron and copper redox proteins Both Cu and Fe proteins are capable of supporting a wide range of similar biological functions

4. Iron-heme proteins

5. 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)

6. 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

7. 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

8. 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

9. 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

10. 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)

11. Iron-sulfur proteins

12. Functions of Iron-Sulfur Centers • photosynthesis • cell respiration • nitrogen fixation • metabolism of hydrogen • NO2 and SO3 oxidation & reduction • redox catalysis

13. 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

14. Iron-sulfur clustersrepresentative examples The charges represent the most common oxidation states for each cluster

15. Iron-sulfur proteins

16. Iron-sulfur proteinsspectroscopic properties Changes in the environment around each type of cluster are used to fine-tune the redox potentials

17. Rubredoxin1Fe-0S cluster High-spin, ferric iron by EPR and Mossbauer spectroscopy Redox potential of rubredoxins range from -50 to +50 mV

18. Ferredoxin2Fe-2S cluster High-spin, ferric iron magnetically coupled (EPR silent)

19. 4Fe-4S Cluster distorted cubic shape bound to the protein through four cysteine thiolates

20. 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

21. 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

22. 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)

23. non-heme iron proteins

24. 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

25. Ribonucleotide reductase Bimetallic center with bridging glutamate and oxygen ligands

26. 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)

27. 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)

28. 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

29. Copper redox proteins

30. Copper Centers in Proteins coordination geometry structure and function

31. 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

32. 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

33. 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

34. 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)

35. 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)

36. 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

37. 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

38. 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

39. Complex redox proteins

40. 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

41. Photosynthetic Reaction Center There are multiple electron carriers that will funnel electrons into the active site

42. 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

43. 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

44. 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

45. Nitrogenasestructure This enzyme uses two different types of subunits with different metal cofactors to catalyze nitrogen fixation Mo-Fe protein Fe protein

46. 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

47. Sulfite reductase How are 6 electrons accommodated in the enzyme ? The enzyme has two separate metal cofactors

48. 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)

49. 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

50. 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)