Thiolate Spin Population of Type I Copper in Azurin Derived from 33S Hyperfine Coupling.

The electron transfer mediating properties of type I copper proteins stem from the intricate ligand coordination sphere of the Cu ion in their active site. These redox properties are in part due to unusual cysteine thiol coordination, which forms a highly covalent copper-sulfur (Cu-S) bond. The structure and electronic properties of type I copper have been the subject of many experimental and theoretical studies. The measurement of spin delocalization of the Cu(II) unpaired electron to neighboring ligands provides an elegant experimental way to probe the fine details of the electronic structure of type I copper. To date, the crucial parameter of electron delocalization to the sulfur atom of the cysteine ligand has not been directly determined experimentally. We have prepared 33S-enriched azurin and carried out W-band (95 GHz) electron paramagnetic resonance (EPR) and electron-electron double resonance detected NMR (EDNMR) measurements and, for the first time, recorded the 33S nuclear frequencies, from which the hyperfine coupling and the spin population on the sulfur of the thiolate ligand were derived. The overlapping 33S and 14N EDNMR signals were resolved using a recently introduced two-dimensional correlation technique, 2D-EDNMR. The 33S hyperfine tensor was determined by simulations of the EDNMR spectra using 33S hyperfine and quadrupolar tensors predicted by QM/MM DFT calculations as starting points for a manual spectral fit procedure. To reach a reasonable agreement with the experimental spectra, the 33S hyperfine principal value, Az, and one of the corresponding Euler angles had to be modified. The final values obtained gave an experimentally determined sulfur spin population of 29.8 ± 0.7%, significantly improving the wide range of 29-62% reported in the literature. Our direct, experimentally derived value now provides an important constraint for further theoretical work aimed at unravelling the unique electronic properties of this site.

Binuclear Cu(A) Formation in Biosynthetic Models of Cu(A) in Azurin Proceeds via a Novel Cu(Cys)2His Mononuclear Copper Intermediate.

Cu(A) is a binuclear electron transfer (ET) center found in cytochrome c oxidases (CcOs), nitrous oxide reductases (N2ORs), and nitric oxide reductase (NOR). In these proteins, the Cu(A) centers facilitate efficient ET (kET > 104s⁻¹) under low thermodynamic driving forces (10-90 mV). While the structure and functional properties of Cu(A) are well understood, a detailed mechanism of the incorporation of copper into the protein and the identity of the intermediates formed during the Cu(A) maturation process are still lacking. Previous studies of the Cu(A) assembly mechanism in vitro using a biosynthetic model Cu(A) center in azurin (Cu(A)Az) identified a novel intermediate X (Ix) during reconstitution of the binuclear site. However, because of the instability of Ix and the coexistence of other Cu centers, such as Cu(A)' and type 1 copper centers, the identity of this intermediate could not be established. Here, we report the mechanism of Cu(A) assembly using variants of Glu114XCuAAz (X = Gly, Ala, Leu, or Gln), the backbone carbonyl of which acts as a ligand to the Cu(A) site, with a major focus on characterization of the novel intermediate Ix. We show that Cu(A) assembly in these variants proceeds through several types of Cu centers, such as mononuclear red type 2 Cu, the novel intermediate Ix, and blue type 1 Cu. Our results show that the backbone flexibility of the Glu114 residue is an important factor in determining the rates of T2Cu → Ix formation, suggesting that Cu(A) formation is facilitated by swinging of the ligand loop, which internalizes the T2Cu capture complex to the protein interior. The kinetic data further suggest that the nature of the Glu114 side chain influences the time scales on which these intermediates are formed, the wavelengths of the absorption peaks, and how cleanly one intermediate is converted to another. Through careful understanding of these mechanisms and optimization of the conditions, we have obtained Ix in ∼80-85% population in these variants, which allowed us to employ ultraviolet-visible, electron paramagnetic resonance, and extended X-ray absorption fine structure spectroscopic techniques to identify the Ix as a mononuclear Cu(Cys)(2)(His) complex. Because some of the intermediates have been proposed to be involved in the assembly of native Cu(A), these results shed light on the structural features of the important intermediates and mechanism of Cu(A) formation.

Genetic and biochemical investigations of the role of MamP in redox control of iron biomineralization in Magnetospirillum magneticum.

Magnetotactic bacteria have evolved complex subcellular machinery to construct linear chains of magnetite nanocrystals that allow the host cell to sense direction. Each mixed-valent iron nanoparticle is mineralized from soluble iron within a membrane-encapsulated vesicle termed the magnetosome, which serves as a specialized compartment that regulates the iron, redox, and pH environment of the growing mineral. To dissect the biological components that control this process, we have carried out a genetic and biochemical study of proteins proposed to function in iron mineralization. In this study, we show that the redox sites of c-type cytochromes of the Magnetospirillum magneticum AMB-1 magnetosome island, MamP and MamT, are essential to their physiological function and that ablation of one or both heme motifs leads to loss of function, suggesting that their ability to carry out redox chemistry in vivo is important. We also develop a method to heterologously express fully heme-loaded MamP from AMB-1 for in vitro biochemical studies, which show that its Fe(III)-Fe(II) redox couple is set at an unusual potential (-89 ± 11 mV) compared with other related cytochromes involved in iron reduction or oxidation. Despite its low reduction potential, it remains competent to oxidize Fe(II) to Fe(III) and mineralize iron to produce mixed-valent iron oxides. Finally, in vitro mineralization experiments suggest that Mms mineral-templating peptides from AMB-1 can modulate the iron redox chemistry of MamP.

Correlating nuclear frequencies by two-dimensional ELDOR-detected NMR spectroscopy.

ELDOR (Electron Double Resonance)-detected NMR (EDNMR) is a pulse EPR experiment that is used to measure the transition frequencies of nuclear spins coupled to electron spins. These frequencies are further used to determine hyperfine and quadrupolar couplings, which are signatures of the electronic and spatial structures of paramagnetic centers. In recent years, EDNMR has been shown to be particularly useful at high fields/high frequencies, such as W-band (∼95 GHz, ∼3.5 T), for low γ quadrupolar nuclei. Although at high fields the nuclear Larmor frequencies are usually well resolved, the limited resolution of EDNMR still remains a major concern. In this work we introduce a two dimensional, triple resonance, correlation experiment based on the EDNMR pulse sequence, which we term 2D-EDNMR. This experiment allows circumventing the resolution limitation by spreading the signals in two dimensions and the observed correlations help in the assignment of the signals. First we demonstrate the utility of the 2D-EDNMR experiment on a nitroxide spin label, where we observe correlations between (14)N nuclear frequencies. Negative cross-peaks appear between lines belonging to different MS electron spin manifolds. We resolved two independent correlation patterns for nuclear frequencies arising from the EPR transitions corresponding to the (14)N mI=0 and mI=-1 nuclear spin states, which severely overlap in the one dimensional EDNMR spectrum. The observed correlations could be accounted for by considering changes in the populations of energy levels that S=1/2, I=1 spin systems undergo during the pulse sequence. In addition to these negative cross-peaks, positive cross-peaks appear as well. We present a theoretical model based on the Liouville equation and use it to calculate the time evolution of populations of the various energy levels during the 2D-EDNMR experiment and generated simulated 2D-EDMR spectra. These calculations show that the positive cross-peaks appear due to off resonance effects and/or nuclear relaxation effects. These results suggest that the 2D-EDNMR experiment can be also useful for relaxation pathway studies. Finally we present preliminary results demonstrating that 2D-EDNMR can resolve overlapping (33)S and (14)N signals of type 1 Cu(II) center in (33)S enriched Azurin.

Axial interactions in the mixed-valent CuA active site and role of the axial methionine in electron transfer.

Within Cu-containing electron transfer active sites, the role of the axial ligand in type 1 sites is well defined, yet its role in the binuclear mixed-valent CuA sites is less clear. Recently, the mutation of the axial Met to Leu in a CuA site engineered into azurin (CuA Az) was found to have a limited effect on E(0) relative to this mutation in blue copper (BC). Detailed low-temperature absorption and magnetic circular dichroism, resonance Raman, and electron paramagnetic resonance studies on CuA Az (WT) and its M123X (X = Q, L, H) axial ligand variants indicated stronger axial ligation in M123L/H. Spectroscopically validated density functional theory calculations show that the smaller ΔE(0) is attributed to H2O coordination to the Cu center in the M123L mutant in CuA but not in the equivalent BC variant. The comparable stabilization energy of the oxidized over the reduced state in CuA and BC (CuA ∼ 180 mV; BC ∼ 250 mV) indicates that the S(Met) influences E(0) similarly in both. Electron delocalization over two Cu centers in CuA was found to minimize the Jahn-Teller distortion induced by the axial Met ligand and lower the inner-sphere reorganization energy. The Cu-S(Met) bond in oxidized CuA is weak (5.2 kcal/mol) but energetically similar to that of BC, which demonstrates that the protein matrix also serves an entatic role in keeping the Met bound to the active site to tune down E(0) while maintaining a low reorganization energy required for rapid electron transfer under physiological conditions.

Kinetics of copper incorporation into a biosynthetic purple Cu(A) azurin: characterization of red, blue, and a new intermediate species.

Evolutionary links between type 1 blue copper (T1 Cu), type 2 red copper (T2 Cu), and purple Cu(A) cupredoxins have been proposed, but the structural features and mechanism responsible for such links as well as for assembly of Cu(A) sites in vivo are poorly understood, even though recent evidence demonstrated that the Cu(II) oxidation state plays an important role in this process. In this study, we examined the kinetics of Cu(II) incorporation into the Cu(A) site of a biosynthetic Cu(A) model, Cu(A) azurin (Cu(A)Az) and found that both T1 Cu and T2 Cu intermediates form on the path to final Cu(A) reconstitution in a pH-dependent manner, with slower kinetics and greater accumulation of the intermediates as the pH is raised from 5.0 to 7.0. While these results are similar to those observed previously in the native Cu(A) center of nitrous oxide reductase, the faster kinetics of copper incorporation into Cu(A)Az allowed us to use lower copper equivalents to reveal a new pathway of copper incorporation, including a novel intermediate that has not been reported in cupredoxins before, with intense electronic absorption maxima at ~410 and 760 nm. We discovered that this new intermediate underwent reduction to Cu(I), and proposed that it is a Cu(II)-dithiolate species. Oxygen-dependence studies demonstrated that the T1 Cu species only formed in the presence of molecular oxygen, suggesting the T1 Cu intermediate is a one-electron oxidation product of a Cu(I) species. By studying Cu(A)Az variants where the Cys and His ligands are mutated, we have identified the T2 Cu intermediate as a capture complex with Cys116 and the T1 Cu intermediate as a complex with Cys112 and His120. These results led to a unified mechanism of copper incorporation and new insights regarding the evolutionary link between all cupredoxin sites as well as the in vivo assembly of Cu(A) centers.

Rationally tuning the reduction potential of a single cupredoxin beyond the natural range.

Redox processes are at the heart of numerous functions in chemistry and biology, from long-range electron transfer in photosynthesis and respiration to catalysis in industrial and fuel cell research. These functions are accomplished in nature by only a limited number of redox-active agents. A long-standing issue in these fields is how redox potentials are fine-tuned over a broad range with little change to the redox-active site or electron-transfer properties. Resolving this issue will not only advance our fundamental understanding of the roles of long-range, non-covalent interactions in redox processes, but also allow for design of redox-active proteins having tailor-made redox potentials for applications such as artificial photosynthetic centres or fuel cell catalysts for energy conversion. Here we show that two important secondary coordination sphere interactions, hydrophobicity and hydrogen-bonding, are capable of tuning the reduction potential of the cupredoxin azurin over a 700 mV range, surpassing the highest and lowest reduction potentials reported for any mononuclear cupredoxin, without perturbing the metal binding site beyond what is typical for the cupredoxin family of proteins. We also demonstrate that the effects of individual structural features are additive and that redox potential tuning of azurin is now predictable across the full range of cupredoxin potentials.

Experimental evidence for a link among cupredoxins: red, blue, and purple copper transformations in nitrous oxide reductase.

The cupredoxin fold is an important motif in numerous proteins that are central to several critical cellular processes ranging from aerobic and anaerobic respiration to catalysis and iron homeostasis. Three types of copper sites have been found to date within cupredoxin folds: blue type 1 (T1) copper, red type 2 (T2) copper, and purple Cu(A). Although as much as 90% sequence difference has been observed among some members of this superfamily of proteins that span several kingdoms, sequence alignment and phylogenic trees strongly suggest an evolutionary link and common ancestry. However, experimental evidence for such a link has been lacking. We report herein the observation of pH-dependent transformation between blue T1 copper, red T2 copper, and the native purple Cu(A) centers of nitrous oxide reductase (N2OR) from Paracoccus denitrificans. The blue and red copper centers form initially before they are transformed into purple Cu(A) center. This transformation process is pH-dependent, with lower pH resulting in fewer trapped T1 and T2 coppers and faster transition to purple Cu(A). These observations suggest that the purple Cu(A) site contains the essential elements of T1 and T2 copper centers and that the Cu(A) center is preferentially formed at low pH. Therefore, this work provides an underlying link between the various cupredoxin copper sites and possible experimental evidence in vitro for the evolutionary relationship between the cupredoxin proteins. The findings also lend physiological relevance to cupredoxin site biosynthesis.