Mechanisms of Redox-Coupled Proton Transfer in Proteins:  Role of the Proximal Proline in Reactions of the [3Fe-4S] Cluster in Azotobacter vinelandii Ferredoxin I^,

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.

Investigating the function of [2Fe–2S] cluster N1a, the off-pathway cluster in complex I, by manipulating its reduction potential

“…The valine-to-proline substitution replaces a backbone secondary amide, which is close to the cluster and so may form a hydrogen bond to it, with a tertiary amide. It may also affect the protein conformation; replacing a proline residue adjacent to the [3Fe–4S] cluster in Azotobacter vinelandii ferredoxin I caused an additional water molecule to enter the structure, increasing the cluster potential by a similar amount to observed in the present study [43]. …”

Conserved Hydrogen Bonding Networks of MitoNEET Tune Fe-S Cluster Binding and Structural Stability

“…Additional hydrogen bonding networks in the interior of the protein and at the dimer interface, including Arg-73 and His-58 may also be involved in long-range PCET. In the case of some clusters, direct protonation at a μ-sulfido ligand is possible, as described for the [3Fe-4S] cluster of Azotobacter vinlandeii Ferredoxin I (AvFdI) (35). …”

Direct Electrochemistry of Redox Enzymes as a Tool for Mechanistic Studies

“…This suggests that, by examining how the voltammetry depends on pH and scan rate, it is possible to measure the rate of (de)protonation of the center that gives rise to the peak, and indeed this strategy was extensively used for determining the rates and the mechanism of proton transfer to and from the buried [3Fe4S] +/0 cluster in AvFdI. [137][138][139] This work was summarized in recent reviews (see e.g. refs 52 and 53).…”