Outer-sphere contributions to the electronic structure of type zero copper proteins.

Bioinorganic canon states that active-site thiolate coordination promotes rapid electron transfer (ET) to and from type 1 copper proteins. In recent work, we have found that copper ET sites in proteins also can be constructed without thiolate ligation (called "type zero" sites). Here we report multifrequency electron paramagnetic resonance (EPR), magnetic circular dichroism (MCD), and nuclear magnetic resonance (NMR) spectroscopic data together with density functional theory (DFT) and spectroscopy-oriented configuration interaction (SORCI) calculations for type zero Pseudomonas aeruginosa azurin variants. Wild-type (type 1) and type zero copper centers experience virtually identical ligand fields. Moreover, O-donor covalency is enhanced in type zero centers relative that in the C112D (type 2) protein. At the same time, N-donor covalency is reduced in a similar fashion to type 1 centers. QM/MM and SORCI calculations show that the electronic structures of type zero and type 2 are intimately linked to the orientation and coordination mode of the carboxylate ligand, which in turn is influenced by outer-sphere hydrogen bonding.

Spin delocalization over type zero copper.

Hard-ligand, high-potential copper sites have been characterized in double mutants of Pseudomonas aeruginosa azurin (C112D/M121X (X = L, F, I)). These sites feature a small A(zz)(Cu) splitting in the EPR spectrum together with enhanced electron transfer activity. Due to these unique properties, these constructs have been called "type zero" copper sites. In contrast, the single mutant, C112D, features a large A(zz)(Cu) value characteristic of the typical type 2 Cu(II). In general, A(zz)(Cu) comprises contributions from Fermi contact, spin dipolar, and orbital dipolar terms. In order to understand the origin of the low A(zz)(Cu) value of type zero Cu(II), we explored in detail its degree of covalency, as manifested by spin delocalization over its ligands, which affects A(zz)(Cu) through the Fermi contact and spin dipolar contributions. This was achieved by the application of several complementary EPR hyperfine spectroscopic techniques at X- and W-band (∼9.5 and 95 GHz, respectively) frequencies to map the ligand hyperfine couplings. Our results show that spin delocalization over the ligands in type zero Cu(II) is different from that of type 2 Cu(II) in the single C112D mutant. The (14)N hyperfine couplings of the coordinated histidine nitrogens are smaller by about 25-40%, whereas that of the (13)C carboxylate of D112 is about 50% larger. From this comparison, we concluded that the spin delocalization of type zero copper over its ligands is not dramatically larger than in type 2 C112D. Therefore, the reduced A(zz)(Cu) value of type zero Cu(II) is largely attributable to an increased orbital dipolar contribution that is related to its larger g(zz) value, as a consequence of the distorted tetrahedral geometry. The increased spin delocalization over the D112 carboxylate in type zero mutants compared to type 2 C112D suggests that electron transfer paths involving this residue are enhanced.

Electron transfer reactivity of type zero Pseudomonas aeruginosa azurin.

Type zero copper is a hard-ligand analogue of the classical type 1 or blue site in copper proteins that function as electron transfer (ET) agents in photosynthesis and other biological processes. The EPR spectroscopic features of type zero Cu(II) are very similar to those of blue copper, although lacking the deep blue color, due to the absence of thiolate ligation. We have measured the rates of intramolecular ET from the pulse radiolytically generated C3-C26 disulfide radical anion to the Cu(II) in both type zero C112D/M121L and type 2 C112D Pseudomonas aeruginosa azurins in pH 7.0 aqueous solutions between 8 and 45 °C. We also have obtained rate/temperature (10-30 °C) profiles for ET reactions between these mutants and the wild-type azurin. Analysis of the rates and activation parameters for both intramolecular and intermolecular ET reactions indicates that the type zero copper reorganization energy falls in a range (0.9-1.1 eV) slightly above that for type 1 (0.7-0.8 eV), but substantially smaller than that for type 2 (>2 eV), consistent with XAS and EXAFS data that reveal minimal type zero site reorientation during redox cycling.

Outer-sphere effects on reduction potentials of copper sites in proteins: the curious case of high potential type 2 C112D/M121E Pseudomonas aeruginosa azurin.

Redox and spectroscopic (electronic absorption, multifrequency electron paramagnetic resonance (EPR), and X-ray absorption) properties together with X-ray crystal structures are reported for the type 2 Cu(II) C112D/M121E variant of Pseudomonas aeruginosa azurin. The results suggest that Cu(II) is constrained from interaction with the proximal glutamate; this structural frustration implies a "rack" mechanism for the 290 mV (vs NHE) reduction potential measured at neutral pH. At high pH (∼9), hydrogen bonding in the outer coordination sphere is perturbed to allow axial glutamate ligation to Cu(II), with a decrease in potential to 119 mV. These results highlight the role played by outer-sphere interactions, and the structural constraints they impose, in determining the redox behavior of transition metal protein cofactors.

High-potential C112D/M121X (X = M, E, H, L) Pseudomonas aeruginosa azurins.

Site-directed mutagenesis of Pseudomonas aeruginosa azurin C112D at the M121 position has afforded a series of proteins with elevated Cu(II/I) reduction potentials relative to the Cu(II) aquo ion. The high potential and low axial hyperfine splitting (Cu(II) electron paramagnetic resonance A( parallel)) of the C112D/M121L protein are remarkably similar to features normally associated with type 1 copper centers.

Electron Tunneling through Pseudomonas aeruginosa Azurins on SAM Gold Electrodes.

Robust voltammetric responses were obtained for wild-type and Y72F/H83Q/Q107H/Y108F azurins adsorbed on CH(3)(CH(2))(n)SH:HO(CH(2))(m)SH (n=m=4,6,8,11; n=13,15 m=11) self-assembled monolayer (SAM) gold electrodes in acidic solution (pH 4.6) at high ionic strengths. Electron-transfer (ET) rates do not vary substantially with ionic strength, suggesting that the SAM methyl headgroup binds to azurin by hydrophobic interactions. The voltammetric responses for both proteins at higher pH values (>4.6 to 11) also were strong. A binding model in which the SAM hydroxyl headgroup interacts with the Asn47 carboxamide accounts for the relatively strong coupling to the copper center that can be inferred from the ET rates. Of particular interest is the finding that rate constants for electron tunneling through n = 8, 13 SAMs are higher at pH 11 than those at pH 4.6, possibly owing to enhanced coupling of the SAM to Asn 47 caused by deprotonation of nearby surface residues.

Probing folded and unfolded states of outer membrane protein a with steady-state and time-resolved tryptophan fluorescence.

Steady-state and time-resolved fluorescence measurements on each of five native tryptophan residues in full-length and truncated variants of E. coli outer-membrane protein A (OmpA) have been made in folded and denatured states. Tryptophan singlet excited-state lifetimes are multiexponential and vary among the residues. In addition, substantial increases in excited-state lifetimes accompany OmpA folding, with longer lifetimes in micelles than in phospholipid bilayers. This finding suggests that the Trp environments of OmpA folded in micelles and phospholipid bilayers are different. Measurements of Trp fluorescence decay kinetics with full-length OmpA folded in brominated lipid vesicles reveal that W102 is the most distant fluorophore from the hydrocarbon core, while W7 is the closest. Steady-state and time-resolved polarized fluorescence measurements indicate reduced Trp mobility when OmpA is folded in a micelle, and even lower mobility when the protein is folded in a bilayer. The fluorescence properties of truncated OmpA, in which the soluble periplasmic domain is removed, only modestly differ from those of the full-length form, suggesting similar folded structures for the two forms under these conditions.

Cloning, heterologous expression, and characterization of recombinant class II cytochromes c from Rhodopseudomonas palustris.

The cytochrome (cyt) c', cyt c(556), and cyt c(2) genes from Rhodopseudomonas palustris have been cloned; recombinant cyt c' and cyt c(556) have been expressed, purified, and characterized. Unlike mitochondrial cyt c, these two proteins are structurally similar to cyt b(562), in which the heme is embedded in a four-helix bundle. The hemes in both recombinant proteins form covalent thioether links to two Cys residues. UV/vis spectra of the Fe(II) and Fe(III) states of the recombinant cyts are identical with those of the corresponding native proteins. Equilibrium unfolding measurements in guanidine hydrochloride solutions confirm that native Fe(II)-cyt c(556) is more stable than the corresponding state of Fe(III)-cyt c(556) (DeltaDeltaG(f)(o) =22 kJ/mol).

X-ray Absorption Spectra of the Oxidized and Reduced Forms of C112D Azurin from Pseudomonas aeruginosa.

The oxidized and reduced forms of a mutant of Pseudomonas aeruginosa azurin, in which the Cys112 has been replaced by an aspartate, have been studied by X-ray absorption spectroscopy. It is well established that the characteristic approximately 600 nm absorption feature of blue copper proteins is due to the S(Cys112) 3ppi --> Cu 3d(x)()()2(-)(y)()()2 charge-transfer transition. While other mutagenesis studies have involved the creation of an artificial blue copper site, the present work involves a mutant in which the native blue copper site has been destroyed, thus serving as a direct probe of the importance of the copper-thiolate bond to the spectroscopy, active site structure, and electron-transfer function of azurin. Of particular interest is the dramatic decrease in electron-transfer rates, both electron self-exchange (k(ese) approximately 10(5) M(-)(1) s(-)(1) wild-type azurin vs k(ese) approximately 20 M(-)(1) s(-)(1) C112D azurin) and intramolecular electron transfer to ruthenium-labeled sites (k(et) approximately 10(6) s(-)(1) wild-type azurin vs k(et)

An outer-sphere hydrogen-bond network constrains copper coordination in blue proteins.

In azurins and other blue copper proteins with relatively low reduction potentials (E(0) [Cu(II)/Cu(I)]<400 mV vs. normal hydrogen electrode), the folded polypeptide framework constrains both copper(II) and copper(I) in such a way as to tune the reduction potentials to values that differ greatly from those for most copper complexes. Largely conserved networks of hydrogen bonds organize and lock the rest of the folded protein structure to a loop that contains three of the ligands to copper. Changes in hydrogen bonds that allow copper(I) to revert more closely to its preferred geometry [relative to the copper(II) geometry] accordingly lead to an increase in E(0). This paper reports mutations in the ligand loop of amicyanin from P. denitrificans that relax the constraints on ligation for copper(I) and significantly raise E(0) for these mutants (for example 415+/-4 mV) relative to that of the native amicyanin (265+/-4 mV). These mutations also shift the pK(a) of a ligand histidine to below 5 relative to 7.0 in the wild type.

Inner- and outer-sphere metal coordination in blue copper proteins.

Blue copper proteins (BCPs) comprise classic cases of Nature's profound control over the electronic structures and chemical reactivity of transition metal ions. Early studies of BCPs focused on their inner coordination spheres, that is, residues that directly coordinate Cu. Equally important are the electronic and geometric perturbations to these ligands provided by the outer coordination sphere. In this tribute to Hans Freeman, we review investigations that have advanced the understanding of how inner-sphere and outer-sphere coordination affects biological Cu properties.

Type-zero copper proteins.

Many proteins contain copper in a range of coordination environments, where it has various biological roles, such as transferring electrons or activating dioxygen. These copper sites can be classified by their function or spectroscopic properties. Those with a single copper atom are either type 1, with an intense absorption band near 600 nm, or type 2, with weak absorption in the visible region. We have built a novel copper(II) binding site within structurally modified Pseudomonas aeruginosa azurins that does not resemble either existing type, which we therefore call 'type zero'. X-ray crystallographic analysis shows that these sites adopt distorted tetrahedral geometries, with an unusually short Cu­O (G45 carbonyl) bond. Relatively weak absorption near 800 nm and narrow parallel hyperfine splittings in electron paramagnetic resonance spectra are the spectroscopic signatures of type zero copper. Cyclic voltammetric experiments demonstrate that the electron transfer reactivities of type-zero azurins are enhanced relative to that of the corresponding type 2 (C112D) protein.

Tryptophan-accelerated electron flow through proteins.

Energy flow in biological structures often requires submillisecond charge transport over long molecular distances. Kinetics modeling suggests that charge-transfer rates can be greatly enhanced by multistep electron tunneling in which redox-active amino acid side chains act as intermediate donors or acceptors. We report transient optical and infrared spectroscopic experiments that quantify the extent to which an intervening tryptophan residue can facilitate electron transfer between distant metal redox centers in a mutant Pseudomonas aeruginosa azurin. Cu(I) oxidation by a photoexcited Re(I)-diimine at position 124 on a histidine(124)-glycine(123)-tryptophan(122)-methionine(121) beta strand occurs in a few nanoseconds, fully two orders of magnitude faster than documented for single-step electron tunneling at a 19 angstrom donor-acceptor distance.

Excited-state dynamics of structurally characterized [ReI(CO)3(phen)(HisX)]+ (X = 83, 109) Pseudomonas aeruginosa azurins in aqueous solution.

The triplet metal-to-ligand charge transfer ((3)MLCT) dynamics of two structurally characterized Re(I)(CO)(3)(phen)(HisX)-modified (phen = 1,10-phenanthroline; X = 83, 109) Pseudomonas aeruginosa azurins have been investigated by picosecond time-resolved infrared (TRIR) spectroscopy in aqueous (D(2)O) solution. The (3)MLCT relaxation dynamics exhibited by the two Re(I)-azurins are very different from those of the sensitizer [Re(I)(CO)(3)(phen)(im)](+) (im = imidazole). Whereas the Re(I)(CO)(3) intramolecular vibrational relaxation in Re(I)(CO)(3)(phen)(HisX)Az (4 ps) is similar to that of [Re(I)(CO)(3)(phen)(im)](+) (2 ps), the medium relaxation is much slower ( approximately 250 vs 9.5 ps); the 250-ps relaxation is attributable to reorientation of D(2)O molecules as well as structural reorganization of the rhenium chromophore and nearby polar amino acids in each of the modified proteins.

Mimicking protein-protein electron transfer: voltammetry of Pseudomonas aeruginosa azurin and the Thermus thermophilus Cu(A) domain at omega-derivatized self-assembled-monolayer gold electrodes.

Well-defined voltammetric responses of redox proteins with acidic-to-neutral pI values have been obtained on pure alkanethiol as well as on mixed self-assembled-monolayer (SAM) omega-derivatized alkanethiol/gold bead electrodes. Both azurin (P. aeruginosa) (pI = 5.6) and subunit II (Cu(A) domain) of ba(3)-type cytochrome c oxidase (T. thermophilus) (pI = 6.0) exhibit optimal voltammetric responses on 1:1 mixtures of [H(3)C(CH(2))(n)()SH + HO(CH(2))(n)()SH] SAMs. The electron transfer (ET) rate vs distance behavior of azurin and Cu(A) is independent of the omega-derivatized alkanethiol SAM headgroups. Strikingly, only wild-type azurin and mutants containing Trp48 give voltammetric responses: based on modeling, we suggest that electronic coupling with the SAM headgroup (H(3)C- and/or HO-) occurs at the Asn47 side chain carbonyl oxygen and that an Asn47-Cys112 hydrogen bond promotes intramolecular ET to the copper. Inspection of models also indicates that the Cu(A) domain of ba(3)-type cytochrome c oxidase is coupled to the SAM headgroup (H(3)C- and/or HO-) near the main chain carbonyl oxygen of Cys153 and that Phe88 (analogous to Trp143 in subunit II of cytochrome c oxidase from R. sphaeroides) is not involved in the dominant tunneling pathway. Our work suggests that hydrogen bonds from hydroxyl or other proton-donor groups to carbonyl oxygens potentially can facilitate intermolecular ET between physiological redox partners.