Redox thermodynamics of mutant forms of the rubredoxin from Clostridiumpasteurianum: identification of a stable Fe(III)(S-Cys)3(OH) centre in the C6S mutant.
Redox thermodynamic data provide a detailed insight into control of the reduction potential E degrees' of the [Fe(S-Cys)4] site in rubredoxin. Mutant forms were studied in which specific structural changes were made in both the primary and secondary coordination spheres. Those changes have been probed by resonance Raman spectroscopy. The decrease of approximately 200 mV in E degrees' observed for the [Fe(S-Cys)3(O-Ser)]-/2- couples in the surface ligand mutants C9S and C42S is essentially enthalpic in origin and associated with the substitution of ligand thiolate by ligand olate. However, the pH dependence of the potentials below characteristic pKa(red) approximately equals 7 is an entropic contribution, plausibly associated with increased conformational flexibility induced by a longer Fe(II)-O(H)-Ser bond in the reduced form. The presence of a second surface Ser ligand in the new double mutant protein C9S/C42S affects the enthalpic term primarily for pH>pKa(red) > or = 9.3, but for pH<pKa(red) the entropic term again becomes significant. The available data for the internal ligand mutant C39S appear to follow those for the surface ligand mutants. A longer Fe(III)-O-Ser link in the oxidized form is expected from structural considerations and a smaller decrease of approximately 100 mV in E degrees' is observed for this particular [Fe(S-Cys)3(O-Ser)]/2- couple. The reduced form is particularly susceptible to hydrolysis with consequent irreversible electrochemistry, an apparent consequence of an even longer Fe(II)-O(H)-Ser bond. The second internal ligand mutant C6S exhibits starkly different behaviour. The cause was traced to the presence of a Fe(III)(S-Cys)3(OH)- centre in the oxidized form. Resonance Raman spectroscopy on 54Fe-, H2 18O- and D2O-enriched samples supports the presence of an iron hydroxyl group that donates a hydrogen bond to the OH group of the free S6 side chain and/or a cluster of bound water molecules. The thermodynamic parameters can then be rationalized in terms of the following processes: pH>pKa approximately 9: [Fe(III)(S-Cys)3(OH)]- + e- --> [Fe(II)(S-Cys)3(OH)]2-. pH<pKa: [Fe(III)(S-Cys)3(OH)]- +H+ e- --> [Fe(II)(S-Cys)3(OH2)]-.
Xiao
Z
Z
School of Chemistry, University of Melbourne, Parkville, Victoria, Australia. z.xiao@unimelb.edu.au
Gardner
A R
AR
Cross
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M
Maes
E M
EM
Czernuszewicz
R S
RS
Sola
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Wedd
A G
AG
D003013:Clostridium / Q000737:chemistry* / Q000378:metabolism; D003545:Cysteine / Q000235:genetics; D008958:Models, Molecular; D009154:Mutation; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012416:Rubredoxins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D012694:Serine / Q000235:genetics; D013816:Thermodynamics
D011994:Recombinant Proteins; D012416:Rubredoxins; D012694:Serine; D003545:Cysteine