Understanding the mechanism of short-range electron transfer using an immobilized cupredoxin.

The hydrophobic patch of azurin (AZ) from Pseudomonas aeruginosa is an important recognition surface for electron transfer (ET) reactions. The influence of changing the size of this region, by mutating the C-terminal copper-binding loop, on the ET reactivity of AZ adsorbed on gold electrodes modified with alkanethiol self-assembled monolayers (SAMs) has been studied. The distance-dependence of ET kinetics measured by cyclic voltammetry using SAMs of variable chain length, demonstrates that the activation barrier for short-range ET is dominated by the dynamics of molecular rearrangements accompanying ET at the AZ-SAM interface. These include internal electric field-dependent low-amplitude protein motions and the reorganization of interfacial water molecules, but not protein reorientation. Interfacial molecular dynamics also control the kinetics of short-range ET for electrostatically and covalently immobilized cytochrome c. This mechanism therefore may be utilized for short-distance ET irrespective of the type of metal center, the surface electrostatic potential, and the nature of the protein-SAM interaction.

Cloning, expression, and physicochemical characterization of a new diheme cytochrome c from Shewanella baltica OS155.

The 16-kDa diheme cytochrome c from the bacterium Shewanella baltica OS155 (Sb-DHC) was cloned and expressed in Escherichia coli and investigated through UV-vis, magnetic circular dichroism, and (1)H NMR spectroscopies and protein voltammetry. The model structure was obtained by means of comparative modeling using the X-ray structure of Rhodobacter sphaeroides diheme cytochrome c (Rs-DHC) (with a 37% pairwise sequence identity) as a template. Sb-DHC folds into two distinct domains, each containing one heme center with a bis-His axial ligation. Both secondary and tertiary structures of the N-terminal domain resemble those of class I cytochrome c, displaying three α-helices and a compact overall folding. The C-terminal domain is less helical than the corresponding domain of Rs-DHC. The two heme groups are bridged by Tyr26 in correspondence with the shortest edge-to-edge distance, a feature which would facilitate fast internal electron transfer. The electronic properties of the two prosthetic centers are equivalent and sensitive to two acid-base equilibria with pK (a) values of approximately 2.4 and 5, likely corresponding to protonation and detachment of the axial His ligands from the heme iron and a pH-linked conformational change of the protein, respectively. Reduction potentials of -0.144 and -0.257 V (vs. the standard hydrogen electrode), were determined for the C- and N-terminal heme groups, respectively. An approach based on the extended Debye-Hückel equation was applied for the first time to a two-centered metalloprotein and was found to reproduce successfully the ionic strength dependence of E°'.

Metal-binding loop length is a determinant of the pKa of a histidine ligand at a type 1 copper site.

The type 1 copper site of a cupredoxin involves coordination by cysteine, histidine, and methionine residues from a single loop. Dissociation and protonation of the histidine ligand on this loop is observed in only certain reduced cupredoxins and can regulate electron-transfer reactivity. This effect is introduced in azurin (AZ) (the wild-type protein has an estimated pKa of <2) by mutating the native copper-binding loop (C(112)TFPGH(117)SALM(121), ligands numbered). In this work, we have investigated the influence of loop length alone on histidine ligand protonation by determining the pKa value in AZ variants with ligand-containing polyalanine loops of different length. Crystal structures of the Cu(I)-variant with the loop sequence C(112)AAH(115)AAM(118) (AZ2A2A) demonstrate that at pH 4.2 His115 is protonated and no longer coordinated, and the imidazole ring is rotated by 180°. The influence of pH on the reduction potential allows a pKa of 5.2 ± 0.1 for His115 in Cu(I)-AZ2A2A to be determined. In the reduced AZ variants in which the loop sequences C(112)AAAAH(117)AAAM(121) (AZ4A3A) and C(112)AAAAH(117)AAAAM(122) (AZ4A4A) have been introduced, pKa values of 4.5 ± 0.1 and 4.4 ± 0.1, respectively, are obtained for the His117 ligand. Consistent with these data, the crystal structure of Cu(I)-AZ4A4A at pH 5.3 shows no sign of His117 protonation (crystals were unstable at lower pH values). The loop length range studied matches that which occurs naturally and these investigations indicate that length alone can alter the pKa of the coordinating histidine by approximately 1 pH unit. The pKa for this histidine ligand varies in native cupredoxins by >5 pH units. Other structural and electronic features, governed primarily by the second-coordination sphere, to which the ligand-binding loop is a major contributor, also alter this important feature. A longer ligand-containing loop made of residues whose side chains are larger and more complex than a methyl group increases the second coordination sphere providing additional scope for tuning the pKa of the histidine ligand and other active site properties.

The impact of urea-induced unfolding on the redox process of immobilised cytochrome c.

We have studied the effect of urea-induced unfolding on the electron transfer process of yeast iso-1-cytochrome c and its mutant K72AK73AK79A adsorbed on electrodes coated by mixed 11-mercapto-1-undecanoic acid/11-mercapto-1-undecanol self-assembled monolayers. Electrochemical measurements, complemented by surface enhanced resonance Raman studies, indicate two distinct states of the adsorbed proteins that mainly differ with respect to the ligation pattern of the haem. The native state, in which the haem is axially coordinated by Met80 and His18, displays a reduction potential that slightly shifts to negative values with increasing urea concentration. At urea concentrations higher than 6 M, a second state prevails in which the Met80 ligand is replaced by an additional histidine residue. This structural change in the haem pocket is associated with an approximately 0.4 V shift of the reduction potential to negative values. These two states were found for both the wild-type protein and the mutant in which lysine residues 72, 73 and 79 had been substituted by alanines. The analysis of the reduction potentials, the reaction enthalpies and entropies as well as the rate constants indicates that these three lysine residues have an important effect on stabilising the protein structure in the adsorbed state and facilitating the electron transfer dynamics.

Redox and electrocatalytic properties of mimochrome VI, a synthetic heme peptide adsorbed on gold.

Mimochrome VI (MC-VI) is a synthetic heme peptide containing a helix-heme-helix sandwich motif designed to reproduce the catalytic activity of heme oxidases. The thermodynamics of Fe(III) to Fe(II) reduction and the kinetics of the electron-transfer process for MC-VI immobilized through hydrophobic interactions on a gold electrode coated with a nonpolar SAM of decane-1-thiol have been determined through cyclic voltammetry. Immobilization slightly affects the reduction potential of MC-VI, which under these conditions electrocatalytically turns over molecular oxygen. This work sets the premise for the exploitation of totally synthetic mimochrome-modified electrode surfaces for clinical and pharmaceutical biosensing.

Immobilized unfolded cytochrome c acts as a catalyst for dioxygen reduction.

Unfolding turns immobilized cytochrome c into a His-His ligated form endowed with catalytic activity towards O(2), which is absent in the native protein. Dioxygen could be used by naturally occurring unfolded cytochrome c as a substrate for the production of partially reduced oxygen species (PROS) contributing to the cell oxidative stress.

Heterogeneous electron transfer of a two-centered heme protein: redox and electrocatalytic properties of surface-immobilized cytochrome C(4).

The recombinant diheme cytochrome c(4) from the psycrophilic bacterium Pseudoalteromonas haloplanktis TAC 125 and its Met64Ala and Met164Ala variants, which feature a hydroxide ion axially bound to the heme iron at the N- and C-terminal domains, respectively, were found to exchange electrons efficiently with a gold electrode coated with a SAM of 11-mercapto-1-undecanoic acid. The mutation-induced removal of the redox equivalence of the two heme groups and changes in the net charge of the protein lobes yield two-centered protein systems with unprecedented properties in the electrode-immobilized state. The heterogeneous and intraheme electron transfer processes were characterized for these species in which the high- and low-potential heme groups are swapped over in the bilobal protein framework and experience a constrained (M64A) and unconstrained (M164A) orientation toward the electrode. The reduction thermodynamics for the native and mutated hemes were measured for the first time for a diheme cytochrome c. In the diffusing regime, they reproduce closely those for the corresponding centers in single-heme class-I cytochromes c, despite the low sequence identity. Larger differences are observed in the thermodynamics of the immobilized species and in the heterogeneous electron transfer rate constants. T-dependent kinetic measurements show that the proteins are positioned approximately 7 A from the HOOC-terminated SAM-coated electrode. Protein-electrode orientation and efficient intraheme ET enable the His,OH(-)-ligated heme A of the immobilized Met64Ala variant to carry out the reductive electrocatalysis of molecular oxygen. This system therefore constitutes a novel two-centered heme-based biocatalytic interface to be exploited for "third-generation" amperometric biosensing.