R Camba

University of Oxford, Oxford, ENG, United Kingdom

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Publications (14)138.19 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: The 7Fe ferredoxin from Azotobacter vinelandii (AvFdI) contains a [3Fe-4S](+/0) cluster that binds a single proton in its reduced level. Although the cluster is buried, and therefore inaccessible to solvent, proton transfer from solvent to the cluster is fast. The kinetics and energetics of the coupled electron-proton transfer reaction at the cluster have been analyzed in detail by protein-film voltammetry, to reveal that proton transfer is mediated by the mobile carboxylate of an adjacent surface residue, aspartate-15, the pK of which is sensitive to the charge on the cluster. This paper examines the role of a nearby proline residue, proline-50, in proton transfer and its coupling to electron transfer. In the P50A and P50G mutants, a water molecule has entered the cluster binding region; it is hydrogen bonded to the backbone amide of residue-50 and to the Asp-15 carboxylate, and it is approximately 4 A from the closest sulfur atom of the cluster. Despite the water molecule linking the cluster more directly to the solvent, proton transfer is not accelerated. A detailed analysis reveals that Asp-15 remains a central part of the mechanism. However, the electrostatic coupling between cluster and carboxylate is almost completely quenched, so that cluster reduction no longer induces such a favorable shift in the carboxylate pK, and protonation of the base no longer induces a significant shift in the pK of the cluster. The electrostatic coupling is crucial for maintaining the efficiency of proton transfer both to and from the cluster, over a range of pH values.
    Biochemistry 10/2003; 42(36):10589-99. · 3.38 Impact Factor
  • Journal of Inorganic Biochemistry - J INORG BIOCHEM. 01/2003; 96(1):96-96.
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    ABSTRACT: The redox properties of the blue copper protein amicyanin have been studied with slow and fast scan protein-film cyclic voltammetry. At slow scan rates, which reveal the thermodynamics of the redox reactions, the reduction potential of amicyanin depends on pH in a sigmoidal manner, and the data can be analysed in terms of electron transfer being coupled to a single protonatable group with pKa(red)=6.3 and pKa(ox) < or = 3.2 at 22 degrees C. Voltammetry at higher scan rates reveals the kinetics and shows that the low-pH reduced form of amicyanin is not oxidised directly; instead, oxidation occurs only after conversion to the high-pH form. Simulations show that this conversion, which gates the electron transfer, occurs with a rate constant >750 s-1 at 25 degrees C. In order to decrease the rate of the coupled reaction, the experiments were performed at 0 degrees C, at which the rate constant for this conversion was determined to be 35 +/- 20 s-1. Together with evidence from NMR, the results lead to a mechanism involving protonation and dissociation of the copper coordinating histidine-96 in the reduced form.
    JBIC Journal of Biological Inorganic Chemistry 01/2002; 7(1-2):94-100. · 3.35 Impact Factor
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    ABSTRACT: Interconversion between [4Fe-4S] cubane and [3Fe-4S] cuboidal states represents one of the simplest structural changes an iron-sulphur cluster can undertake. This reaction is implicated in oxidative damage and in modulation of the activity and regulation of certain enzymes, and it is therefore important to understand the factors governing cluster stability and the processes that activate cluster conversion. In the present study, protein film voltammetry has been used to induce and monitor the oxidative conversion of [4Fe-4S] into [3Fe-4S] clusters in different variants of Azotobacter vinelandii ferredoxin I (AvFdI; the 8Fe form of the native protein), and DeltaThr(14)/DeltaAsp(15), Thr(14)-->Cys (T14C) and C42D mutants. The electrochemical results have been correlated with the differing oxygen sensitivities of [4Fe-4S] clusters, and comparisons have been drawn with other ferredoxins (Desulfovibrio africanus FdIII, Clostridium pasteurianum Fd, Thauera aromatica Fd and Pyrococcus furiosus Fd). In contrast with high-potential iron-sulphur proteins (HiPIPs) for which the oxidized species [4Fe-4S](3+) is inert to degradation and can be isolated, the hypervalent state in these ferredoxins (most obviously the 3+ level) is very labile, and the reduction potential at which this is formed is a key factor in determining the cluster's resistance to oxidative damage.
    Biochemical Journal 12/2001; 360(Pt 3):717-26. · 4.65 Impact Factor
  • Biochemical Journal - BIOCHEM J. 01/2001; 360(3).
  • R Camba
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    ABSTRACT: Traditionally, libraries attempted to prove their effectiveness by reporting the number of resources the library bought or subscribed to, of instructional sessions taught and of reference questions answered, among other statistics. However, libraries are increasingly expected to document student achievement using outcomes assessment. After struggling with outcomes assessment at our own institution for several years, we have found that the most effective way to handle program-level and classroom-level outcomes assessment is to create manageable, realistic assessment tools. In this paper, we describe two assessment tools that have worked for us: a brief survey given to a large number of students and an in-depth, multipart tool used with a limited number of library instruction sessions.
    Nature 09/2000; 406(6795):461. · 38.60 Impact Factor
  • R Camba, F A Armstrong
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    ABSTRACT: Rapid responses of biological [4Fe-4S] clusters to conditions of oxidative stress have been studied by protein-film voltammetry by using precise pulses of electrode potential to trigger reactions. Investigations with Clostridium pasteurianum 8Fe ferredoxin exploit the fact that [3Fe-4S] clusters display a characteristic pattern of voltammetric signals, so that their appearance and disappearance after an oxidative pulse can be tracked unambiguously under electrochemical control. Adsorbed to monolayer coverage at a graphite electrode, the protein initially shows a strong signal (B') at -0.36 V vs standard hydrogen electrode due to two [4Fe-4S](2+/+) clusters at similar potentials. Short square pulses (0.1-5 s) to potentials in the range 0.5-0.9 V cause extensive loss of B', and new signals appear (A'and C') that arise from [3Fe-4S] species (+/0 and 0/2- couples). The A' and B' intensities quantify transformations which are induced by the pulse and which occur subsequently when more reducing conditions are restored. Optimal [3Fe-4S] formation (in excess over [4Fe-4S]) is achieved with a 3-s pulse to 0.7 V, following which there is rapid partial recovery to yield a 1:1 3Fe:4Fe ratio, consistent with 7Fe protein. Thus, a 6Fe protein is formed, but one of the clusters is rapidly repaired. The [3Fe-4S]:[4Fe-4S] ratio follows a bell-shaped curve spanning the same potential range that defines complete loss of signals, while double-pulse experiments show that [3Fe-4S](+) resists further oxidative damage. Oxidative disassembly involves successive one-electron oxidations of [4Fe-4S] (i.e., 2+ --> 3+ --> 4+), with [3Fe-4S](+) being a relatively stable byproduct, that is, not an intermediate. Disassembly of [3Fe-4S] in the 7Fe protein continues after reducing conditions are restored, with lifetimes depending on oxidation level; thus 1+ (most stable) > 0 > 2-. In the presence of Fe(2+), the 0 level is stabilized by conversion back to [4Fe-4S](2+/+). By pulsing in the presence of Zn(2+), the [3Fe-4S] clusters that are formed are trapped rapidly as their Zn adducts.
    Biochemistry 08/2000; 39(34):10587-98. · 3.38 Impact Factor
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    ABSTRACT: The basis of the chemiosmotic theory is that energy from light or respiration is used to generate a trans-membrane proton gradient. This is largely achieved by membrane-spanning enzymes known as 'proton pumps. There is intense interest in experiments which reveal, at the molecular level, how protons are drawn through proteins. Here we report the mechanism, at atomic resolution, for a single long-range electron-coupled proton transfer. In Azotobacter vinelandii ferredoxin I, reduction of a buried iron-sulphur cluster draws in a solvent proton, whereas re-oxidation is 'gated' by proton release to the solvent. Studies of this 'proton-transferring module' by fast-scan protein film voltammetry, high-resolution crystallography, site-directed mutagenesis and molecular dynamics, reveal that proton transfer is exquisitely sensitive to the position and pK of a single amino acid. The proton is delivered through the protein matrix by rapid penetrative excursions of the side-chain carboxylate of a surface residue (Asp 15), whose pK shifts in response to the electrostatic charge on the iron-sulphur cluster. Our analysis defines the structural, dynamic and energetic requirements for proton courier groups in redox-driven proton-pumping enzymes.
    Nature 07/2000; 405(6788):814-7. · 38.60 Impact Factor
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    ABSTRACT: A wealth of information on the reactions of redox-active sites in proteins can be obtained by voltammetric studies in which the protein sample is arranged as a layer on an electrode surface. By carrying out cyclic voltammetry over a wide range of scan rates and exploiting the ability to poise or pulse the electrode potential between cycles, data are obtained that are conveniently (albeit simplistically) analysed in terms of plots of peak potentials against scan rate. A simple reversible electron-transfer process gives rise to a 'trumpet'-shaped plot because the oxidation and reduction peaks separate increasingly at high scan rate; the electrochemical kinetics are then determined by fitting to Butler-Volmer or Marcus models. Much more interesting though are the ways in which this 'trumpet plot' is altered, often dramatically, when electron transfer is coupled to biologically important processes such as proton transfer, ligand exchange, or a change in conformation. It is then possible to derive particularly detailed information on the kinetics, energetics and mechanism of reactions that may not revealed clearly or even at all by other methods. In order to interpret the voltammetry of coupled systems, it is important to be able to define 'ideal behaviour' for systems that are expected to show simple and uncoupled electron transfer. Accordingly, this paper describes results we have obtained for several proteins that are expected to show such behaviour, and compares these results with theoretical predictions.
    Faraday Discussions 02/2000; · 3.82 Impact Factor
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    ABSTRACT: A study of the structure and redox properties of the copper site in azurins by means of EXAFS, NMR, redox titrations, potentiometry, equilibrium cyclic voltammetry and rapid scan voltammetry on protein films is reported. The results are discussed in light of existing theories on structure and function of type-1 copper sites. The exit and entry of electrons take place through the C-terminal histidine ligand of the copper. The hydrophobic patch through which this residue penetrates the protein surface plays an important role in partner docking (cf. The rim of the porphyrin ring sticking through the surface of the cytochromes-c). We find no experimental evidence for strain around the metal site. The active centre is able to maintain ET activity even in the presence of fairly gross disturbances of the site structure. The analysis of the thermodynamics of the redox reaction shows that the protein matrix and the solvent play an important role in 'tuning' the redox potential around a "design" value of around 300 mV at room temperature. The metal site appears "designed" to stabilise the Cu(II) instead of the Cu(I) form. The remarkable evolutionary success of the blue copper proteins is ascribed to the sturdy overall beta-sandwich structure of the protein in combination with a metal site that is structurally adaptable because three of its four ligands are located on a loop. The electronic "gate" that occurs in the middle of a hydrophobic patch allows for fine tuning of the docking patch for recognition purposes.
    Faraday Discussions 02/2000; · 3.82 Impact Factor
  • Journal of The American Chemical Society - J AM CHEM SOC. 01/2000; 122(27):6494-6495.
  • Journal of the American Chemical Society. 01/2000; 122(49):12186-12194.
  • [Show abstract] [Hide abstract]
    ABSTRACT: The basis of the chemiosmotic theory is that energy from light or respiration is used to generate a trans-membrane proton gradient. This is largely achieved by membrane-spanning enzymes known as `proton pumps'. There is intense interest in experiments which reveal, at the molecular level, how protons are drawn through proteins.Here we report the mechanism, at atomic resolution, for a single long-range electron-coupled proton transfer. In Azotobacter vinelandii ferredoxin I, reduction of a buried iron-sulphur cluster draws in a solvent proton, whereas re-oxidation is `gated' by proton release to the solvent. Studies of this `proton-transferring module' by fast-scan protein film voltammetry, high-resolution crystallography, site-directed mutagenesis and molecular dynamics, reveal that proton transfer is exquisitely sensitive to the position and pK of a single amino acid. The proton is delivered through the protein matrix by rapid penetrative excursions of the side-chain carboxylate of a surface residue (Asp15), whose pK shifts in response to the electrostatic charge on the iron-sulphur cluster. Our analysis defines the structural, dynamic and energetic requirements for proton courier groups in redox-driven proton-pumping enzymes.
    Nature 01/2000; 407. · 38.60 Impact Factor
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Publication Stats

204 Citations
138.19 Total Impact Points

Institutions

  • 2000–2003
    • University of Oxford
      • • Department of Chemistry
      • • Inorganic Chemistry Laboratory
      Oxford, ENG, United Kingdom
    • University of California, Irvine
      • Department of Molecular Biology and Biochemistry
      Irvine, CA, United States