Fraser A. Armstrong

Dynamic electrochemistry of iron−sulfur proteins

Publisher Summary This chapter discusses the contribution that can be made by dynamic electrochemical methods toward the often-difficult task of defining reactions of Fe–S clusters in a protein molecule. Despite the relatively modest amount of material published to date, these reports show that such an approach, when combined with appropriate spectroscopic techniques, can yield valuable insight. The adsorbed film studies of Dimorphognathus africanus Fd III described in the chapter illustrate a new strategy for studying proteins with very reactive Fe–S clusters. The approach combines high sensitivity, accuracy, and the ability to study reactions under strict conditions of applied potential. Equilibrium and kinetic data may be derived with trace amounts of protein, and verified by the preparation of appropriate spectroscopic samples. From the results of spectroscopic studies on the products of parallel transformations carried out in solution, it is demonstrated in the chapter that the voltammetric interrogation of Fe–S proteins in an adsorbed film can provide an acceptably valid reflection of the chemistry of free molecules. A useful trailblazing capability is thus provided to give guidance and to complement the techniques aimed at detailed structural characterization.

Discovery of a Novel Ferredoxin from Azotobacter vinelandii Containing Two [4Fe-4S] Clusters with Widely Differing and Very Negative Reduction Potentials

Ferredoxins that contain 2[4Fe-4S]2+/+ clusters can be divided into two classes. The “clostridial-type” ferredoxins have two Cys-Xaa-Xaa-Cys-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Cys-Pro motifs. The “chromatium-type” ferredoxins have one motif of that type and one more unusual Cys-Xaa-Xaa-Cys-Xaa7–9-Cys-Xaa-Xaa-Xaa-Cys-Pro motif.Here we report the purification of a novel ferredoxin (FdIII) from Azotobacter vinelandii which brings to 12 the number of small [Fe-S] proteins that have now been reported from this organism. NH2-terminal sequencing of the first 56 amino acid residues shows that FdIII is a chromatium-type ferredoxin with 77% identity and 88% similarity to Chromatium vinosumferredoxin. Studies of the purified protein by matrix-assisted laser desorption ionization–time of flight mass spectroscopy, iron analysis, absorption, circular dichroism, and electron paramagnetic resonance spectroscopies show that FdIII contains 2[4Fe-4S]2+/+clusters in a 9,220-Da polypeptide. All 2[4Fe-4S]2+/+ferredoxins that have been studied to date, including C. vinosum ferredoxin, are reported to have extremely similar or identical reduction potentials for the two clusters. In contrast, electrochemical characterization of FdIII clearly establishes that the two [4Fe-4S]2+/+ clusters have very different and highly negative reduction potentials of −486 mV and −644 mVversus the standard hydrogen electrode.

Classification of fumarate reductases and succinate dehydrogenases based upon their contrasting behaviour in the reduced benzylviologen/fumarate assay

Reduction of fumarate by soluble beef heart succinate dehydrogenase has been shown previously by voltammetry to become increasingly retarded as the potential is lowered below a threshold potential of −80 mV at pH 7.5. The behaviour resembles that of a tunnel diode, an electronic device exhibiting the property of negative resistance. The enzyme thus acts to oppose fumarate reduction under conditions of high thermodynamic driving force. We now provide independent evidence for this phenomenon from spectrophotometric kinetic assays. With reduced benzylviologen as electron donor, we have studied the reduction of fumarate catalysed by various enzymes classified either as succinate dehydrogenases or fumarate reductases. For succinate dehydrogenases, the rate increases as the concentration of reduced dye (driving force) decreases during the reaction. In contrast, authentic fumarate reductases of anaerobic cells (and ‘succinate dehydrogenase’ from Bacillus subtilis) neither exhibit the electrochemical effect nor deviate from simple kinetic behaviour in the cuvette assay. The ‘tunnel‐diode’ effect may thus represent an evolutionary adaptation to aerobic metabolism.

Alteration of the Reduction Potential of the [4Fe-4S]2+/+ Cluster of Azotobacter vinelandii Ferredoxin I

The [4Fe-4S]2+/+ cluster ofAzotobacter vinelandii ferredoxin I (FdI) has an unusually low reduction potential (E 0′) relative to other structurally similar ferredoxins. Previous attempts to raise thatE 0′ by modification of surface charged residues were unsuccessful. In this study mutants were designed to alter theE 0′ by substitution of polar residues for nonpolar residues near the cluster and by modification of backbone amides. Three FdI variants, P21G, I40N, and I40Q, were purified and characterized, and electrochemical E 0′measurements show that all had altered E 0′relative to native FdI. For P21G FdI and I40Q FdI, theE 0′ increased by +42 and +53 mV, respectively validating the importance of dipole orientation in control ofE 0′. Protein Dipole Langevin Dipole calculations based on models for those variants accurately predicted the direction of the change in E 0′ while overestimating the magnitude. For I40N FdI, initial calculations based on the model predicted a +168 mV change in E 0′while a −33 mV change was observed. The x-ray structure of that variant, which was determined to 2.8 Å, revealed a number of changes in backbone and side chain dipole orientation and in solvent accessibility, that were not predicted by the model and that were likely to influence E 0′. Subsequent Protein Dipole Langevin Dipole calculations (using the actual I40N x-ray structures) did quite accurately predict the observed change inE 0′.

Structure of C42D Azotobacter vinelandii FdI A Cys-X-X-Asp-X-X-Cys MOTIF LIGATES AN AIR-STABLE [4Fe-4S]2+/+ CLUSTER

All naturally occurring ferredoxins that have Cys-X-X-Asp-X-X-Cys motifs contain [4Fe-4S]2+/+ clusters that can be easily and reversibly converted to [3Fe-4S]+/0 clusters. In contrast, ferredoxins with unmodified Cys-X-X-Cys-X-X-Cys motifs assemble [4Fe-4S]2+/+ clusters that cannot be easily interconverted with [3Fe-4S]+/0 clusters. In this study we changed the central cysteine of the Cys39-X-X-Cys42-X-X-Cys45of Azotobacter vinelandii FdI, which coordinates its [4Fe-4S]2+/+ cluster, into an aspartate. UV-visible, EPR, and CD spectroscopies, metal analysis, and x-ray crystallography show that, like native FdI, aerobically purified C42D FdI is a seven-iron protein retaining its [4Fe-4S]2+/+ cluster with monodentate aspartate ligation to one iron. Unlike known clusters of this type the reduced [4Fe-4S]+ cluster of C42D FdI exhibits only an S = 1/2 EPR with no higher spin signals detected. The cluster shows only a minor change in reduction potential relative to the native protein. All attempts to convert the cluster to a 3Fe cluster using conventional methods of oxygen or ferricyanide oxidation or thiol exchange were not successful. The cluster conversion was ultimately accomplished using a new electrochemical method. Hydrophobic and electrostatic interaction and the lack of Gly residues adjacent to the Asp ligand explain the remarkable stability of this cluster.

Azotobacter vinelandii Ferredoxin I A SEQUENCE AND STRUCTURE COMPARISON APPROACH TO ALTERATION OF [4Fe-4S]2+/+ REDUCTION POTENTIAL

The reduction potential (E 0′) of the [4Fe-4S]2+/+ cluster of Azotobacter vinelandii ferredoxin I (AvFdI) and related ferredoxins is ∼200 mV more negative than the corresponding clusters of Peptostreptococcus asaccharolyticus ferredoxin and related ferredoxins. Previous studies have shown that these differences inE 0′ do not result from the presence or absence of negatively charged surface residues or in differences in the types of hydrophobic residues found close to the [4Fe-4S]2+/+clusters. Recently, a third, quite distinct class of ferredoxins (represented by the structurally characterized Chromatium vinosum ferredoxin) was shown to have a [4Fe-4S]2+/+ cluster with a very negativeE 0′ similar to that of AvFdI. The observation that the sequences and structures surrounding the very negative E 0′ clusters in quite dissimilar proteins were almost identical inspired the construction of three additional mutations in the region of the [4Fe-4S]2+/+cluster of AvFdI. The three mutations, V19E, P47S, and L44S, that incorporated residues found in the higherE 0′ P. asaccharolyticus ferredoxin all led to increases in E 0′ for a total of 130 mV with a 94-mV increase in the case of L44S. The results are interpreted in terms of x-ray structures of the FdI variants and show that the major determinant for the large increase in L44S is the introduction of an OH–S bond between the introduced Ser side chain and the Sγ atom of Cys ligand 42 and an accompanying movement of water.

Iron—sulphur clusters with labile metal ions

A study has been carried out of the redox-linked metal ion uptake processes of the iron-sulphur cluster [3Fe-4S] in the bacterial ferredoxin, Fd III from Desulphovibrio africanus using a combination of electron paramagnetic resonance (EPR) and low-temperature magnetic circular dichroism (MCD) spectroscopy and direct, unmediated electrochemistry of the Fd in a film deposited at a pyrolytic graphite electrode. Reduction of the three-iron cluster is required before a divalent metal ion becomes bound as in the reaction sequence [formula: see text] The redox potentials of these processes and the metal binding constants have been determined. The affinities of the [3Fe-4S]0 cluster for divalent ions lie in the sequence Cd greater than Zn much greater than Fe. In addition, specific binding of a monovalent ion, Thallium(I), is detected for [3Fe-4S]1+ as well as for [3Fe-4S]0. The results provide a clear and quantitative demonstration of the capability of the open triangular tri-mu 2-sulphido face of a [3Fe-4S] cluster to bind a variety of metal ions if the protein environment permits. In each case the entering metal ion is coordinated by at least one additional ligand which may be from solvent (H2O or OH-) or from a protein side chain (e.g., carboxylate from aspartic acid). Hence the [3Fe-4S] core can be a redox-linked sensor of divalent metal ions, Fe(II) or Zn(II), that may trigger conformational change.

Y13C Azotobacter vinelandii Ferredoxin I A DESIGNED [Fe-S] LIGAND MOTIF CONTAINS A CYSTEINE PERSULFIDE

Ferredoxins that contain [4Fe-4S]2+/+ clusters often obtain three of their four cysteine ligands from a highly conserved CysXXCysXXCys sequence motif. Little is known about the in vivo assembly of these clusters and the role that this sequence motif plays in that process. In this study, we have used structure as a guide in attempts to direct the formation of a [4Fe-4S]2+/+ in the [3Fe-4S]+/0location of native (7Fe) Azotobacter vinelandiiferredoxin I (AvFdI) by providing the correct three-dimensional orientation of cysteine ligands without introducing a CysXXCysXXCys motif. Tyr13 ofAvFdI occupies the position of the fourth ligating cysteine in the homologous and structurally characterized 8Fe ferredoxin fromPeptococcus aerogenes and a Y13C variant ofAvFdI could be easily modeled as an 8Fe protein. However, characterization of purified Y13C FdI by UV-visible spectra, circular dichroism, electron paramagnetic resonance spectroscopies, and by x-ray crystallography revealed that the protein failed to use the introduced cysteine as a ligand and retained its [3Fe-4S]+/0cluster. Further, electrochemical characterization showed that the redox potential and pH behavior of the cluster were unaffected by the substitution of Tyr by Cys. Although Y13C FdI is functional in vivo it does differ significantly from native FdI in that it is extremely unstable in the reduced state possibly due to increased solvent exposure of the [3Fe-4S]0 cluster. Surprisingly, the x-ray structure showed that the introduced cysteine was modified to become a persulfide. This modification may have occurred in vivo via the action of NifS, which is known to be expressed under the growth conditions used. It is interesting to note that neither of the two free cysteines present in FdI was modified. Thus, ifNifS is involved in modifying the introduced cysteine there must be specificity to the reaction.