The Putidaredoxin Reductase-Putidaredoxin Electron Transfer Complex

Interaction and electron transfer between putidaredoxin reductase (Pdr) and putidaredoxin (Pdx) fromPseudomonas putidawas studied by molecular modeling, mutagenesis, and stopped flow techniques. Based on the crystal structures of Pdr and Pdx, a complex between the proteins was generated using computer graphics methods. In the model, Pdx is docked above the isoalloxazine ring of FAD of Pdr with the distance between the flavin and [2Fe-2S] of 14.6 Å. This mode of interaction allows Pdx to easily adjust and optimize orientation of its cofactor relative to Pdr. The key residues of Pdx located at the center, Asp38and Trp106, and at the edge of the protein-protein interface, Tyr33and Arg66, were mutated to test the Pdr-Pdx computer model. The Y33F, Y33A, D38N, D38A, R66A, R66E, W106F, W106A, and Δ106 mutations did not affect assembly of the [2Fe-2S] cluster and resulted in a marginal change in the redox potential of Pdx. The electron-accepting ability of Δ106 Pdx was similar to that of the wild-type protein, whereas electron transfer rates from Pdr to other mutants were diminished to various degrees with the smallest and largest effects on the kinetic parameters of the Pdr-to-Pdx electron transfer reaction caused by the Trp106and Tyr33/Arg66substitutions, respectively. Compared with wild-type Pdx, the binding affinity of all studied mutants to Pdr was significantly higher. Experimental results were in agreement with theoretical predictions and suggest that: (i) Pdr-Pdx complex formation is mainly driven by steric complementarity, (ii) bulky side chains of Tyr33, Arg66, and Trp106prevent tight binding of oxidized Pdx and facilitate dissociation of the reduced iron-sulfur protein from Pdr, and (iii) transfer of an electron from FAD to [2Fe-2S] can occur with various orientations between the cofactors through multiple electron transfer pathways that do not involve Trp106but are likely to include Asp38and Cys39.

Download Full-text

The Hydrophobic Recognition Site Formed by Residues PsaA-Trp651and PsaB-Trp627of Photosystem I inChlamydomonas reinhardtiiConfers Distinct Selectivity for Binding of Plastocyanin and Cytochromec6

Journal of Biological Chemistry ◽

10.1074/jbc.m313986200 ◽

2004 ◽

Vol 279 (19) ◽

pp. 20009-20017 ◽

Cited By ~ 34

Author(s):

Frederik Sommer ◽

Friedel Drepper ◽

Wolfgang Haehnel ◽

Michael Hippler

Keyword(s):

Electron Transfer ◽

Dissociation Constant ◽

Photosystem I ◽

Tight Binding ◽

Recognition Site ◽

Site Directed Mutagenesis ◽

Tryptophan Residue ◽

Wild Type ◽

Differential Selectivity ◽

Electron Transfer Complex

On the lumenal side of photosystem I (PSI), each of the two large core subunits, PsaA and PsaB, expose a conserved tryptophan residue to the surface. PsaB-Trp627is part of the hydrophobic recognition site that is essential for tight binding of the two electron donors plastocyanin and cytochromec6to the donor side of PSI (Sommer, F., Drepper, F., and Hippler, M. (2002)J. Biol. Chem.277, 6573–6581). To examine the function of PsaA-Trp651in binding and electron transfer of both donors to PSI, we generated the mutants PsaA-W651F and PsaA-W651S by site-directed mutagenesis and biolistic transformation ofChlamydomonas reinhardtii.The protein-protein interaction and the electron transfer between the donors and PSI isolated from the mutants were analyzed by flash absorption spectroscopy. The mutation PsaA-W651F completely abolished the formation of a first order electron transfer complex between plastocyanin (pc) and the altered PSI and increased the dissociation constant for binding of cytochrome (cyt)c6by more than a factor of 10 as compared with wild type. Mutation of PsaA-Trp651to Ser had an even larger impact on the dissociation constant. TheKDvalue increased another 2-fold when the values obtained for the interaction and electron transfer between cytc6and PSI from PsaA-W651S and PsaA-W651F are compared. In contrast, binding and electron transfer of pc to PSI from PsaA-W651S improved as compared with PSI from PsaA-W651F and admitted the formation of an inter-molecular electron transfer complex, resulting in aKDvalue of about 554 μmthat is still five times higher than observed for wild type. These results demonstrate that PsaA-Trp651is, such as PsaB-Trp627, crucial for high affinity binding of pc and cytc6to PSI. Our results also indicate that the highly conserved structural recognition motif that is formed by PsaA-Trp651and PsaB-Trp627confers a differential selectivity in binding of both donors to PSI.

Download Full-text

Flavin radicals, conformational sampling and robust design principles in interprotein electron transfer: the trimethylamine dehydrogenase-electron-transferring flavoprotein complex

Biochemical Society Symposium ◽

10.1042/bss0710001 ◽

2004 ◽

Vol 71 ◽

pp. 1-14

Author(s):

David Leys ◽

Jaswir Basran ◽

François Talfournier ◽

Kamaldeep K. Chohan ◽

Andrew W. Munro ◽

...

Keyword(s):

Electron Transfer ◽

Dimensional Space ◽

Three Dimensional ◽

Kinetic Studies ◽

Molecular Assembly ◽

Conformational Sampling ◽

Trimethylamine Dehydrogenase ◽

Key Residues ◽

Electron Transferring Flavoprotein ◽

Electron Transfer Complex

TMADH (trimethylamine dehydrogenase) is a complex iron-sulphur flavoprotein that forms a soluble electron-transfer complex with ETF (electron-transferring flavoprotein). The mechanism of electron transfer between TMADH and ETF has been studied using stopped-flow kinetic and mutagenesis methods, and more recently by X-ray crystallography. Potentiometric methods have also been used to identify key residues involved in the stabilization of the flavin radical semiquinone species in ETF. These studies have demonstrated a key role for 'conformational sampling' in the electron-transfer complex, facilitated by two-site contact of ETF with TMADH. Exploration of three-dimensional space in the complex allows the FAD of ETF to find conformations compatible with enhanced electronic coupling with the 4Fe-4S centre of TMADH. This mechanism of electron transfer provides for a more robust and accessible design principle for interprotein electron transfer compared with simpler models that invoke the collision of redox partners followed by electron transfer. The structure of the TMADH-ETF complex confirms the role of key residues in electron transfer and molecular assembly, originally suggested from detailed kinetic studies in wild-type and mutant complexes, and from molecular modelling.

Download Full-text

Vanadium nitrogenase of Azotobacter chroococcum. MgATP-dependent electron transfer within the protein complex

Biochemical Journal ◽

10.1042/bj2570789 ◽

1989 ◽

Vol 257 (3) ◽

pp. 789-794 ◽

Cited By ~ 16

Author(s):

R N F Thorneley ◽

N H J Bergström ◽

R R Eady ◽

D J Lowe

Keyword(s):

Electron Transfer ◽

Transfer Reaction ◽

Competitive Inhibitor ◽

Electron Transfer Reaction ◽

Azotobacter Chroococcum ◽

First Order ◽

Fe Protein ◽

Vanadium Nitrogenase ◽

Electron Transfer Complex ◽

Kinetics Of

The kinetics of MgATP-induced electron transfer from the Fe protein (Ac2V) to the VFe protein (AclV) of the vanadium-containing nitrogenase from Azotobacter chroococcum were studied by stopped-flow spectrophotometry at 23 degrees C at pH 7.2. They are very similar to those of the molybdenum nitrogenase of Klebsiella pneumoniae [Thorneley (1975) Biochem. J. 145, 391-396]. Extrapolation of the dependence of kobs. on [MgATP] to infinite MgATP concentration gave k = 46 s-1 for the first-order electron-transfer reaction that occurs with the Ac2V MgATPAclV complex. MgATP binds with an apparent KD = 230 +/- 10 microM and MgADP acts as a competitive inhibitor with Ki = 30 +/- 5 microM. The Fe protein and VFe protein associate with k greater than or equal to 3 x 10(7) M-1.s-1. A comparison of the dependences of kobs. for electron transfer on protein concentrations for the vanadium nitrogenase from A. chroococcum with those for the molybdenum nitrogenase from K. pneumoniae [Lowe & Thorneley (1984) Biochem. J. 224, 895-901] indicates that the proteins of the vanadium nitrogenase system form a weaker electron-transfer complex.

Download Full-text

Structure and function of Aspergillus niger laccase McoG

Biocatalysis ◽

10.1515/boca-2017-0001 ◽

2017 ◽

Vol 3 (1) ◽

pp. 1-21 ◽

Cited By ~ 4

Author(s):

Marta Ferraroni ◽

Adrie H. Westphal ◽

Marco Borsari ◽

Juan Antonio Tamayo-Ramos ◽

Fabrizio Briganti ◽

...

Keyword(s):

Crystal Structure ◽

Electron Transfer ◽

Aspergillus Niger ◽

Structure And Function ◽

Redox Potentials ◽

Wild Type ◽

Multicopper Oxidases ◽

Transfer Rates ◽

The Common ◽

And Function

AbstractThe ascomycete Aspergillus niger produces several multicopper oxidases, but their biocatalytic properties remain largely unknown. Elucidation of the crystal structure of A. niger laccase McoG at 1.7 Å resolution revealed that the C-terminal tail of this glycoprotein blocks the T3 solvent channel and that a peroxide ion bridges the two T3 copper atoms. Remarkably, McoG contains a histidine (His253) instead of the common aspartate or glutamate expected to be involved in catalytic proton transfer with phenolic compounds. The crystal structure of H253D at 1.5 Å resolution resembles the wild type structure. McoG and the H253D, H253A and H253N variants have similar activities with 2,2’-azino-bis(3- ethylbenzothiazoline-6-sulphonic acid or N,N-dimethyl-p-phenylenediamine sulphate. However, the activities of H253A and H253N with 2-amino-4-methylphenol and 2-amino-4-methoxyphenol are strongly reduced compared to that of wild type. The redox potentials and electron transfer rates (k

Download Full-text

Molecular Basis for the Interactions of Human Thioredoxins with Their Respective Reductases

Oxidative Medicine and Cellular Longevity ◽

10.1155/2021/6621292 ◽

2021 ◽

Vol 2021 ◽

pp. 1-17

Author(s):

Md Faruq Hossain ◽

Yana Bodnar ◽

Calvin Klein ◽

Clara Ortegón Salas ◽

Elias S. J. Arnér ◽

...

Keyword(s):

Electron Transfer ◽

Active Site ◽

Molecular Basis ◽

Redox Regulation ◽

Transfer Reaction ◽

Catalytic Efficiency ◽

Electron Transfer Reaction ◽

Wild Type ◽

Electrostatic Properties ◽

Encounter Complex

The mammalian cytosolic thioredoxin (Trx) system consists of Trx1 and its reductase, the NADPH-dependent seleno-enzyme TrxR1. These proteins function as electron donor for metabolic enzymes, for instance in DNA synthesis, and the redox regulation of numerous processes. In this work, we analysed the interactions between these two proteins. We proposed electrostatic complementarity as major force controlling the formation of encounter complexes between the proteins and thus the efficiency of the subsequent electron transfer reaction. If our hypothesis is valid, formation of the encounter complex should be independent of the redox reaction. In fact, we were able to confirm that also a redox inactive mutant of Trx1 lacking both active site cysteinyl residues (C32,35S) binds to TrxR1 in a similar manner and with similar kinetics as the wild-type protein. We have generated a number of mutants with alterations in electrostatic properties and characterised their interaction with TrxR1 in kinetic assays. For human Trx1 and TrxR1, complementary electrostatic surfaces within the area covered in the encounter complex appear to control the affinity of the reductase for its substrate Trx. Electrostatic compatibility was even observed in areas that do not form direct molecular interactions in the encounter complex, and our results suggest that the electrostatic complementarity in these areas influences the catalytic efficiency of the reduction. The human genome encodes ten cytosolic Trx-like or Trx domain-containing proteins. In agreement with our hypothesis, the proteins that have been characterised as TrxR1 substrates also show the highest similarity in their electrostatic properties.

Download Full-text

Evidence for the Head Domain Movement of the Rieske Iron-Sulfur Protein in Electron Transfer Reaction of the Cytochromebc1Complex

Journal of Biological Chemistry ◽

10.1074/jbc.274.11.7146 ◽

1999 ◽

Vol 274 (11) ◽

pp. 7146-7152 ◽

Cited By ~ 63

Author(s):

Hua Tian ◽

Steve White ◽

Linda Yu ◽

Chang-An Yu

Keyword(s):

Electron Transfer ◽

Transfer Reaction ◽

Electron Transfer Reaction ◽

Iron Sulfur ◽

Head Domain ◽

Domain Movement ◽

Sulfur Protein ◽

Iron Sulfur Protein ◽

Rieske Iron Sulfur Protein

Download Full-text

Effects of pH on kinetics of the structural rearrangement that gates the electron-transfer reaction between zinc cytochrome c and plastocyanin: Analysis of protonation states in a diprotein complex

Journal of the Serbian Chemical Society ◽

10.2298/jsc0305327c ◽

2003 ◽

Vol 68 (4-5) ◽

pp. 327-337

Author(s):

Milan Crnogorac ◽

Nenad Kostic

Keyword(s):

Electron Transfer ◽

Cytochrome C ◽

Structural Rearrangement ◽

Electron Transfer Reaction ◽

Salt Bridges ◽

Wild Type ◽

Redox Active ◽

Effects Of Ph ◽

In The Wild ◽

Ph Range

Electron transfer from zinc cytochrome c to copper(II)plastocyanin in the electrostatically- stabilized complex [Crnogorac MM, Shen C, Young S Hansson O, Kostic NM (1996) Biochemistry 35, 16465?74]. We study this rearrangement in four complexes Zncyt/pc(II), which zinc cytochrome c makes with the wild-type form and the single mutants Asp42Asn, Glu59Gln, and Glu60Gln of plastocyanin. The rate constant for the rearrangement, kF differs for the four forms of plastocyanin but is independent of pH from 5.4 to 9.0 in all four cases. That kF is affected by the single mutations but not by pH changes suggests that the residues Asp 42, Glu59, and Glu60 in the wild-type plastocyanin remain deprotonated (i.e., as anions) within the Zncyt/pc(II) complex throughout the pH range examined. This fact agrees with the notion that loss of salt bridges in the initial (redox-inactive) configuration of the complex is compensated by formation of new salt bridges in the rearranged (redox-active) configuration.

Download Full-text