Analysis of resonance Raman data on the blue copper site in pseudoazurin: excited state π and σ charge transfer distortions and their relation to ground state reorganization energy.

The short Cu(2+)-S(Met) bond in pseudoazurin (PAz) results in the presence of two relatively intense S(p)(π) and S(p)(σ) charge transfer (CT) transitions. This has enabled resonance Raman (rR) data to be obtained for each excited state. The rR data show very different intensity distribution patterns for the vibrations in the 300-500 cm(-1) region. Time-dependent density functional theory (TDDFT) calculations have been used to determine that the change in intensity distribution between the S(p)(π) and S(p)(σ) excited states reflects the differential enhancement of S(Cys) backbone modes with Cu-S(Cys)-C(ß) out-of-plane (oop) and in-plane (ip) bend character in their respective potential energy distributions (PEDs). The rR excited state distortions have been related to ground state reorganization energies (λ s) and predict that, in addition to M-L stretches, the Cu-S(Cys)-C(ß) oop bend needs to be considered. DFT calculations predict a large distortion in the Cu-S(Cys)-C(ß) oop bending coordinate upon reduction of a blue copper (BC) site; however, this distortion is not present in the X-ray crystal structures of reduced BC sites. The lack of Cu-S(Cys)-C(ß) oop distortion upon reduction corresponds to a previously unconsidered constraint on the thiolate ligand orientation in the reduced state of BC proteins and can be considered as a contribution to the entatic/rack nature of BC sites.

A combined fluorescence spectroscopic and electrochemical approach for the study of thioredoxins.

A new way to study the electrochemical properties of proteins by coupling front-face fluorescence spectroscopy with an optically transparent thin-layer electrochemical cell is presented. First, the approach was examined on the basis of the redox-dependent conformational changes in tryptophans in cytochrome c, and its redox potential was successfully determined. Second, an electrochemically induced fluorescence analysis of periplasmic thiol-disulfide oxidoreductases SoxS and SoxW was performed. SoxS is essential for maintaining chemotrophic sulfur oxidation of Paracoccus pantotrophus active in vivo, while SoxW is not essential. According to the potentiometric redox titration of tryptophan fluorescence, the midpoint potential of SoxS was -342 ± 8 mV versus the standard hydrogen electrode (SHE') and that of SoxW was -256 ± 10 mV versus the SHE'. The fluorescence properties of the thioredoxins are presented and discussed together with the intrinsic fluorescence contribution of the tyrosines.

The 1.4 A resolution structure of Paracoccus pantotrophus pseudoazurin.

Pseudoazurins are small type 1 copper proteins that are involved in the flow of electrons between various electron donors and acceptors in the bacterial periplasm, mostly under denitrifying conditions. The previously determined structure of Paracoccus pantotrophus pseudoazurin in the oxidized form was improved to a nominal resolution of 1.4 A, with R and R(free) values of 0.188 and 0.206, respectively. This high-resolution structure makes it possible to analyze the interactions between the monomers and the solvent structure in detail. Analysis of the high-resolution structure revealed the structural regions that are responsible for monomer-monomer recognition during dimer formation and for protein-protein interaction and that are important for partner recognition. The pseudoazurin structure was compared with other structures of various type 1 copper proteins and these were grouped into families according to similarities in their secondary structure; this may be useful in the annotation of copper proteins in newly sequenced genomes and in the identification of novel copper proteins.

A variable temperature spectroscopic study on Paracoccuspantotrophus pseudoazurin: protein constraints on the blue Cu site.

The blue or Type 1 (T1) copper site of Paracoccuspantotrophus pseudoazurin exhibits significant absorption intensity in both the 450 and 600 nm regions. These are sigma and pi S(Cys) to Cu(2+) charge transfer (CT) transitions. The temperature dependent absorption, EPR, and resonance Raman (rR) vibrations enhanced by these bands indicate that a single species is present at all temperatures. This contrasts the temperature dependent behavior of the T1 center in nitrite reductase [S. Ghosh, X. Xie, A. Dey, Y. Sun, C. Scholes, E. Solomon, Proc. Natl. Acad. Sci. 106 (2009) 4969-4974] which has a thioether ligand that is unconstrained by the protein. The lack of temperature dependence in the T1 site in pseudoazurin indicates the presence of a protein constraint similar to the blue Cu site in plastocyanin where the thioether ligand is constrained at 2.8 A. However, plastocyanin exhibits only pi CT. This spectral difference between pseudoazurin and plastocyanin reflects a coupled distortion of the site where the axial thioether in pseudoazurin is also constrained, but at a shorter Cu-S(Met) bond length. This leads to an increase in the Cu(2+)-S(Cys) bond length, and the site undergoes a partial tetragonal distortion in pseudoazurin. Thus, its ground state wavefunction has both sigma and pi character in the Cu(2+)-S(Cys) bond.

Coherent Raman detected electron spin resonance spectroscopy of metalloproteins: linking electron spin resonance and magnetic circular dichroism.

The simultaneous excitation of paramagnetic molecules with optical (laser) and microwave radiation in the presence of a magnetic field can cause an amplitude, or phase, modulation of the transmitted light at the microwave frequency. The detection of this modulation indicates the presence of coupled optical and ESR transitions. The phenomenon can be viewed as a coherent Raman effect or, in most cases, as a microwave frequency modulation of the magnetic circular dichroism by the precessing magnetization. By allowing the optical and magnetic properties of a transition metal ion centre to be correlated, it becomes possible to deconvolute the overlapping optical or ESR spectra of multiple centres in a protein or of multiple chemical forms of a particular centre. The same correlation capability also allows the relative orientation of the magnetic and optical anisotropies of each species to be measured, even when the species cannot be obtained in a crystalline form. Such measurements provide constraints on electronic structure calculations. The capabilities of the method are illustrated by data from the dimeric mixed-valence Cu(A) centre of nitrous oxide reductase (N(2)OR) from Paracoccus pantotrophus.

Sulfur oxidation of Paracoccus pantotrophus: the sulfur-binding protein SoxYZ is the target of the periplasmic thiol-disulfide oxidoreductase SoxS.

The periplasmic thiol-disulfide oxidoreductase SoxS is essential for chemotrophic growth of Paracoccus pantotrophus with thiosulfate. To trap its periplasmic partner, the cysteine residues of the CysXaaXaaCys motif of SoxS (11 kDa) were changed to alanine by site-directed mutagenesis. The disrupted soxS gene of the homogenote mutant G OmegaS was complemented with plasmids carrying the mutated soxS[C13A] or soxS[C16A] gene. Strain G OmegaS(pRD179.6[C16A](S)) displayed a marginal thiosulfate-oxidizing activity, suggesting that Cys13(S) binds the target protein. Evidence is presented that SoxS specifically binds SoxY. (i) Immunoblot analysis using non-reducing SDS gel electrophoresis and anti-SoxS and anti-SoxYZ antibodies identified the respective antigens of strain G OmegaS(pRD179.6[C16A](S)) at the 25 kDa position, suggesting an adduct of about 14 kDa, close to the value expected for SoxY migration. (ii) A mutant unable to produce SoxYZ, such as strain G OmegaX(pRD187.7[C16A](S)), did not form a SoxS(C16A) adduct, while addition of homogeneous SoxYZ resulted in the 25 kDa adduct. (iii) The SoxY and SoxZ subunits were distinguished by site-directed mutagenesis of the cysteine residue in SoxZ. SoxYZ(C53S) formed the 25 kDa adduct with SoxS(C16A). These results demonstrate that the target of SoxS is the sulfur-binding protein SoxY of the SoxYZ complex. As SoxYZ is reversibly inactivated, SoxS may activate SoxYZ as a crucial function for chemotrophy of P. pantotrophus.

The periplasmic thioredoxin SoxS plays a key role in activation in vivo of chemotrophic sulfur oxidation of Paracoccus pantotrophus.

The significance of the soxS gene product on chemotrophic sulfur oxidation of Paracoccus pantotrophus was investigated. The thioredoxin SoxS was purified, and the N-terminal amino acid sequence identified SoxS as the soxS gene product. The wild-type formed thiosulfate-oxidizing activity and Sox proteins during mixotrophic growth with succinate plus thiosulfate, while there was no activity, and only traces of Sox proteins, under heterotrophic conditions. The homogenote mutant strain GBOmegaS is unable to express the soxSR genes, of which soxR encodes a transcriptional regulator. Strain GBOmegaS cultivated mixotrophically showed about 22 % of the specific thiosulfate-dependent O(2) uptake rate of the wild-type, and when cultivated heterotrophically it produced 35 % activity. However, under both mixotrophic and heterotrophic conditions, strain GBOmegaS formed Sox proteins essential for sulfur oxidation in vitro at the same high level as the wild-type produced them during mixotrophic growth. Genetic complementation of strain GBOmegaS with soxS restored the activity upon mixotrophic and heterotrophic growth. Chemical complementation by reductants such as L-cysteine, DTT and tris(2-carboxyethyl)phosphine also restored the activity of strain GBOmegaS in the presence of chloramphenicol, which is an inhibitor of de novo protein synthesis. The data demonstrate that SoxS plays a key role in activation of the Sox enzyme system, and this suggests that SoxS is part of a novel type of redox control in P. pantotrophus.

Structure of the cytochrome complex SoxXA of Paracoccus pantotrophus, a heme enzyme initiating chemotrophic sulfur oxidation.

The sulfur-oxidizing enzyme system (Sox) of the chemotroph Paracoccus pantotrophus is composed of several proteins, which together oxidize hydrogen sulfide, sulfur, thiosulfate or sulfite and transfers the gained electrons to the respiratory chain. The hetero-dimeric cytochrome c complex SoxXA functions as heme enzyme and links covalently the sulfur substrate to the thiol of the cysteine-138 residue of the SoxY protein of the SoxYZ complex. Here, we report the crystal structure of the c-type cytochrome complex SoxXA. The structure could be solved by molecular replacement and refined to a resolution of 1.9A identifying the axial heme-iron coordination involving an unusual Cys-251 thiolate of heme2. Distance measurements between the three heme groups provide deeper insight into the electron transport inside SoxXA and merge in a better understanding of the initial step of the aerobic sulfur oxidation process in chemotrophic bacteria.

Sulfur oxidation in Paracoccus pantotrophus: interaction of the sulfur-binding protein SoxYZ with the dimanganese SoxB protein.

The central protein of the sulfur-oxidizing enzyme system of Paracoccus pantotrophus, SoxYZ, formed complexes with subunits associated and covalently bound. In denaturing SDS-polyacrylamide gel electrophoresis (PAGE) SoxY migrated at 12 and SoxZ at 16kDa. SDS-PAGE of homogeneous SoxYZ without reductant separated dimeric complexes of 25, 29, and 32kDa identified by the N-terminal amino acid sequences as SoxY-Y, SoxY-Z, and SoxZ-Z, and subunit cleavage by reduction suggested their linkage via protein disulfide bonds. SoxYZ was reversibly redox active between -0.25 and 0.2V, as monitored by a combined electrochemical and FTIR spectroscopic approach. The dimanganese SoxB protein (58.611Da) converted the covalently linked heterodimer SoxY-Z to SoxYZ with associated subunits which in turn aggregated to the heterotetramer Sox(YZ)(2). This reaction depended on time and the SoxB concentration, and demonstrated the interaction of these two Sox proteins.