Temporal development of protein structure during S100A11 folding and dimerization probed by oxidative labeling and mass spectrometry

Considerable progress in deciphering the mechanisms of protein folding has been made. However, most work in this area has focused on single-chain systems, whereas the majority of proteins are oligomers. The spontaneous assembly of intact multi-subunit systems from disordered building blocks encompasses the formation of intramolecular as well as intermolecular contacts. Both types of interaction affect the solvent accessibility of individual protein segments. This work employs pulsed hydroxyl radical (·OH) labeling for tracking time-dependent accessibility changes during folding and assembly of the S100A11 homodimer. ·OH induces covalent modifications at exposed residues. Structural snapshots are obtained by combining ·OH labeling with rapid mixing and mass spectrometry. The free subunits are found to possess a partially non-native hydrophobic core that prevents subunit association during the initial stages of the reaction. Instead, the protein forms an early (10 ms) monomeric intermediate that exhibits reduced solvent accessibility in regions distant from helices I and IV, which constitute the dimerization interface. Subunit association is complete after 800 ms, although the protein retains significant disorder in helices II and III at this point. Subsequent consolidation of these elements leads to the native state. The experimental strategy used here could become a general tool for deciphering kinetic mechanisms of biomolecular self-assembly processes.     

Kinetic folding mechanism of an integral membrane protein examined by pulsed oxidative labeling and mass spectrometry

We report the application of pulsed oxidative labeling for deciphering the folding mechanism of a membrane protein. SDS-denatured bacteriorhodopsin (BR) was refolded by mixing with bicelles in the presence of free retinal. At various time points (20 ms to 1 day), the protein was exposed to a microsecond ·OH pulse that induces oxidative modifications at solvent-accessible methionine side chains. The extent of labeling was determined by mass spectrometry. These measurements were complemented by stopped-flow spectroscopy. Major time-dependent changes in solvent accessibility were detected for M20 (helix A) and M118 (helix D). Our kinetic data indicate a sequential folding mechanism, consistent with models previously suggested by others on the basis of optical data. Yet, ·OH labeling provides additional structural insights. An initial folding intermediate I(1) gets populated within 20 ms, concomitantly with formation of helix A. Subsequent structural consolidation leads to a transient species I(2). Noncovalent retinal binding to I(2) induces folding of helix D, thereby generating an intermediate I(R). In the absence of retinal, the latter transition does not take place. Hence, formation of helix D depends on retinal binding, whereas this is not the case for helix A. As the cofactor settles deeper into its binding pocket, a final transient species I(R) is generated. This intermediate converts into native BR within minutes by formation of the retinal-K216 Schiff base linkage. The combination of pulsed covalent labeling and optical spectroscopy employed here should also be suitable for exploring the folding mechanisms of other membrane proteins.     

Nitric Oxide Binds to the Proximal Heme Coordination Site of the Ferrocytochrome c/Cardiolipin Complex: FORMATION MECHANISM AND DYNAMICS*

Mammalian mitochondrial cytochrome c interacts with cardiolipin to form a complex (cyt. c/CL) important in apoptosis. Here we show that this interaction leads to structural changes in ferrocytochrome c that leads to an open coordinate site on the central iron, resulting from the dissociation of the intrinsic methionine residue, where NO can rapidly bind (k = 1.2 × 10⁷ m⁻¹ s⁻¹). Accompanying NO binding, the proximal histidine dissociates leaving the heme pentacoordinate, in contrast to the hexacoordinate nitrosyl adducts of native ferrocytochrome c or of the protein in which the coordinating methionine is removed by chemical modification or mutation. We present the results of stopped-flow and photolysis experiments that show that following initial NO binding to the heme, there ensues an unusually complex set of kinetic steps. The spectral changes associated with these kinetic transitions, together with their dependence on NO concentration, have been determined and lead us to conclude that NO binding to cyt. c/CL takes place via an overall scheme comparable to that described for cytochrome c′ and guanylate cyclase, the final product being one in which NO resides on the proximal side of the heme. In addition, novel features not observed before in other heme proteins forming pentacoordinate nitrosyl species, include a high yield of NO escape after dissociation, rapid (<1 ms) dissociation of proximal histidine upon NO binding and its very fast binding (60 ps) after NO dissociation, and the formation of a hexacoordinate intermediate. These features all point at a remarkable mobility of the proximal heme environment induced by cardiolipin.     

Exploring the cooperativity of the fast folding reaction of a small protein using pulsed thiol labeling and mass spectrometry

It has been difficult to obtain directly residue-specific information on side chain packing during a fast (ms) protein folding reaction. Such information is necessary to determine the extent to which structural changes in different parts of the protein molecule are coupled together in defining the cooperativity of the overall folding transition. In this study, structural changes occurring during the major fast folding reaction of the small protein barstar have been characterized at the level of individual residue side chains. A pulsed cysteine labeling methodology has been employed in conjunction with mass spectrometry. This provides, with ms temporal resolution, direct information on structure formation at 10 different locations in barstar during its folding. Cysteine residues located on the surface of native barstar, at four different positions, remain fully solvent-accessible throughout the folding process, indicating the absence of any ephemeral nonnative structure in which these four cysteine residues get transiently buried. For buried cysteine residues, the rates of the change in cysteine-thiol accessibility to rapid chemical labeling by the thiol reagent methyl methanethiosulfonate appear to be dependent upon the location of the cysteine residue in the protein and are different from the rate measured by the change in tryptophan fluorescence. But the rates vary over only a 3-fold range. Nevertheless, a comparison of the kinetics of the change in accessibility of the cysteine 3 thiol with those of the change in the fluorescence of tryptophan 53, as well as of their denaturant dependences, indicates that the major folding reaction comprises more than one step.     

The Determinants of Stability and Folding in Evolutionarily Diverged Cytochromes c

Cytochrome c has served as a paradigm for the study of protein stability, folding, and molecular evolution, but it remains unclear how these aspects of the protein are related. For example, while the bovine and equine cytochromes c are known to have different stabilities, and possibly different folding mechanisms, it is not known how these differences arise from just three amino acid substitutions introduced during divergence. Using site-selectively incorporated carbon-deuterium bonds, we show that like the equine protein, bovine cytochrome c is induced to unfold by guanidine hydrochloride via a stepwise mechanism, but it does not populate an intermediate as is observed with the equine protein. The increased stability also results in more similar free energies of unfolding observed at different sites within the protein, giving the appearance of a more concerted mechanism. Furthermore, we show that the differences in stability and folding appear to result from a single amino acid substitution that stabilizes a helix by allowing for increased solvation of its N-terminus.     

Characterization of quinol-dependent nitric oxide reductase from Geobacillus stearothermophilus: Enzymatic activity and active site structure

Nitric oxide reductase (NOR) catalyzes the reduction of nitric oxide to generate nitrous oxide. We recently reported on the crystal structure of a quinol-dependent NOR (qNOR) from Geobacillus stearothermophilus [Y. Matsumoto, T. Tosha, A.V. Pisliakov, T. Hino, H. Sugimoto, S. Nagano, Y. Sugita and Y. Shiro, Nat. Struct. Mol. Biol. 19 (2012) 238–246], and suggested that a water channel from the cytoplasm, which is not observed in cytochrome c-dependent NOR (cNOR), functions as a pathway transferring catalytic protons. Here, we further investigated the functional and structural properties of qNOR, and compared the findings with those for cNOR. The pH optimum for the enzymatic reaction of qNOR was in the alkaline range, whereas Pseudomonas aeruginosa cNOR showed a higher activity at an acidic pH. The considerably slower reduction rate, and a correlation of the pH dependence for enzymatic activity and the reduction rate suggest that the reduction process is the rate-determining step for the NO reduction by qNOR, while the reduction rate for cNOR was very fast and therefore is unlikely to be the rate-determining step. A close examination of the heme/non-heme iron binuclear center by resonance Raman spectroscopy indicated that qNOR has a more polar environment at the binuclear center compared with cNOR. It is plausible that a water channel enhances the accessibility of the active site to solvent water, creating a more polar environment in qNOR. This structural feature could control certain properties of the active site, such as redox potential, which could explain the different catalytic properties of the two NORs. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

Protein folding mechanisms studied by pulsed oxidative labeling and mass spectrometry

Deciphering the mechanisms of protein folding remains a considerable challenge. In this review we discuss the application of pulsed oxidative labeling for tracking protein structural changes in a time-resolved fashion. Exposure to a microsecond OH pulse at selected time points during folding induces the oxidation of solvent-accessible side chains, whereas buried residues are protected. Oxidative modifications can be detected by mass spectrometry. Folding is associated with dramatic accessibility changes, and therefore this method can provide detailed mechanistic insights. Solvent accessibility patterns are complementary to H/D exchange investigations, which report on the extent of hydrogen bonding. This review highlights the application of pulsed OH labeling to soluble proteins as well as membrane proteins.     

Desolvation and Development of Specific Hydrophobic Core Packing during Im7 Folding

Development of a tightly packed hydrophobic core drives the folding of water-soluble globular proteins and is a key determinant of protein stability. Despite this, there remains much to be learnt about how and when the hydrophobic core becomes desolvated and tightly packed during protein folding. We have used the bacterial immunity protein Im7 to examine the specificity of hydrophobic core packing during folding. This small, four-helix protein has previously been shown to fold via a compact three-helical intermediate state. Here, overpacking substitutions, in which residue side-chain size is increased, were used to examine the specificity and malleability of core packing in the folding intermediate and rate-limiting transition state. In parallel, polar groups were introduced into the Im7 hydrophobic core via Val→Thr or Phe→Tyr substitutions and used to determine the solvation status of core residues at different stages of folding. Over 30 Im7 variants were created allowing both series of substitutions to cover all regions of the protein structure. Φ-value analysis demonstrated that the major changes in Im7 core solvation occur prior to the population of the folding intermediate, with key regions involved in docking of the short helix III remaining solvent-exposed until after the rate-limiting transition state has been traversed. In contrast, overpacking core residues revealed that some regions of the native Im7 core are remarkably malleable to increases in side-chain volume. Overpacking residues in other regions of the Im7 core result in substantial (> 2.5 kJ mol^− 1) destabilisation of the native structure or even prevents efficient folding to the native state. This study provides new insights into Im7 folding; demonstrating that whilst desolvation occurs early during folding, adoption of a specifically packed core is achieved only at the very last step in the folding mechanism.     

Cyanobacterial cytochrome cM: Probing its role as electron donor for CuA of cytochrome c oxidase

It is well known that efficient functioning of photosynthetic (PET) and respiratory electron transport (RET) in cyanobacteria requires the presence of either cytochrome c₆ (Cytc₆) or plastocyanin (PC). By contrast, the interaction of an additional redox carrier, cytochrome c_M (Cytc_M), with either PET or RET is still under discussion. Here, we focus on the (putative) role of Cytc_M in cyanobacterial respiration. It is demonstrated that genes encoding the main terminal oxidase (cytochrome c oxidase, COX) and cytochrome c_M are found in all 44 totally or partially sequenced cyanobacteria (except one strain). In order to check whether Cytc_M can act as electron donor to COX, we investigated the intermolecular electron transfer kinetics between Cytc_M and the soluble Cu_A domain (i.e. the donor binding and electron entry site) of subunit II of COX. Both proteins from Synechocystis PCC6803 were expressed heterologously in E. coli. The forward and the reverse electron transfer reactions were studied yielding apparent bimolecular rate constants of (2.4 ± 0.1) × 10⁵ M^− 1 s^− 1 and (9.6 ± 0.4) × 10³ M^− 1 s^− 1 (5 mM phosphate buffer, pH 7, 50 mM KCl). A comparative analysis with Cytc₆ and PC demonstrates that Cytc_M functions as electron donor to Cu_A as efficiently as Cytc₆ but more efficient than PC. Furthermore, we demonstrate the association of Cytc_M with the cytoplasmic and thylakoid membrane fractions by immunobloting and discuss the potential role of Cytc_M as electron donor for COX under stress conditions.     

Mapping the Structure of an Integral Membrane Protein under Semi-Denaturing Conditions by Laser-Induced Oxidative Labeling and Mass Spectrometry

Little is known about the structural properties of semi-denatured membrane proteins. The current study employs laser-induced oxidative labeling of methionine side chains in combination with electrospray mass spectrometry and optical spectroscopy for gaining insights into the conformation of bacteriorhodopsin (BR) under partially denaturing conditions. The native protein shows extensive oxidation at M32, M68, and M163, which are located in solvent-accessible loops. In contrast, M20 (helix A), M56/60 (helix B), M118 (helix D), M145 (helix E), and M209 (helix G) are strongly protected, consistent with the known protein structure. Exposure of the protein to acidic conditions leads to a labeling pattern very similar to that of the native state. The absence of large-scale conformational changes at low pH is in agreement with recent crystallography data. Solubilization of BR in SDS induces loss of the retinal chromophore concomitant with collapse of the binding pocket, thereby precluding solvent access to the protein interior. Tryptophan fluorescence data confirm the presence of a large protein core that remains protected from water. However, oxidative labeling indicates partial unfolding of helices A and D in SDS. Irreversible thermal denaturation of the protein at 100 °C induces a labeling pattern quite similar to that seen upon SDS exposure. Labeling experiments on refolded bacterioopsin reveal a native-like structure, but with partial unfolding of helix D. Our data suggest that noncovalent contacts with the retinal chromophore in native BR play an important role for the stability of this particular helix. Overall, the present work illustrates the viability of using laser-induced oxidative labeling as a novel tool for characterizing structural changes of membrane proteins in response to alterations of their solvent environment.     

Early Hydrophobic Collapse of α1-Antitrypsin Facilitates Formation of a Metastable State: Insights from Oxidative Labeling and Mass Spectrometry

The biologically active conformation of α₁-antitrypsin (α₁AT) and other serine protease inhibitors represents a metastable state, characterized by an exposed reactive center loop (RCL) that acts as bait for the target enzyme. The protein can also adopt an inactive “latent” conformation that has the RCL inserted as a central strand in β-sheet A. This latent form is thermodynamically more stable than the active conformation. Nonetheless, folding of α₁AT consistently yields the active state. The reasons that the metastable form is kinetically preferred remain controversial. The current work demonstrates that a carefully orchestrated folding mechanism prevents RCL insertion into sheet A. Temporal changes in solvent accessibility during folding are monitored using pulsed oxidative labeling and mass spectrometry. The data obtained in this way complement recent hydrogen/deuterium exchange results. Those hydrogen/deuterium exchange measurements revealed that securing of the RCL by hydrogen bonding of the first β‐strand in sheet C is one factor that favors formation of the active conformation. The oxidative labeling data presented here reveal that this anchoring is preceded by the formation of hydrophobic contacts in a confined region of the protein. This partial collapse sequesters the RCL insertion site early on and is therefore instrumental in steering α₁AT towards its active conformation. RCL anchoring by hydrogen bonding starts to contribute at a later stage. Together, these two factors ensure that formation of the active conformation is kinetically favored. This work demonstrates how the use of complementary labeling techniques can provide insights into the mechanisms of protracted folding reactions.     

Crystal Structure Determination and Functional Characterization of the Metallochaperone SlyD from Thermus thermophilus

SlyD (sensitive to lysis D; product of the slyD gene) is a prolyl isomerase [peptidyl-prolyl cis/trans isomerase (PPIase)] of the FK506 binding protein (FKBP) type with chaperone properties. X-ray structures derived from three different crystal forms reveal that SlyD from Thermus thermophilus consists of two domains representing two functional units. PPIase activity is located in a typical FKBP domain, whereas chaperone function is associated with the autonomously folded insert-in-flap (IF) domain. The two isolated domains are stable and functional in solution, but the presence of the IF domain increases the PPIase catalytic efficiency of the FKBP domain by 2 orders of magnitude, suggesting that the two domains act synergistically to assist the folding of polypeptide chains. The substrate binding surface of SlyD from T. thermophilus was mapped by NMR chemical shift perturbations to hydrophobic residues of the IF domain, which exhibits significantly reduced thermodynamic stability according to NMR hydrogen/deuterium exchange and fluorescence equilibrium transition experiments. Based on structural homologies, we hypothesize that this is due to the absence of a stabilizing β-strand, suggesting in turn a mechanism for chaperone activity by ‘donor-strand complementation.’ Furthermore, we identified a conserved metal (Ni²⁺) binding site at the C-terminal SlyD-specific helical appendix of the FKBP domain, which may play a role in metalloprotein assembly.     

Cytochrome c1 exhibits two binding sites for cytochrome c in plants

In plants, channeling of cytochrome c molecules between complexes III and IV has been purported to shuttle electrons within the supercomplexes instead of carrying electrons by random diffusion across the intermembrane bulk phase. However, the mode plant cytochrome c behaves inside a supercomplex such as the respirasome, formed by complexes I, III and IV, remains obscure from a structural point of view. Here, we report ab-initio Brownian dynamics calculations and nuclear magnetic resonance-driven docking computations showing two binding sites for plant cytochrome c at the head soluble domain of plant cytochrome c₁, namely a non-productive (or distal) site with a long heme-to-heme distance and a functional (or proximal) site with the two heme groups close enough as to allow electron transfer. As inferred from isothermal titration calorimetry experiments, the two binding sites exhibit different equilibrium dissociation constants, for both reduced and oxidized species, that are all within the micromolar range, thus revealing the transient nature of such a respiratory complex. Although the docking of cytochrome c at the distal site occurs at the interface between cytochrome c₁ and the Rieske subunit, it is fully compatible with the complex III structure. In our model, the extra distal site in complex III could indeed facilitate the functional cytochrome c channeling towards complex IV by building a “floating boat bridge” of cytochrome c molecules (between complexes III and IV) in plant respirasome.     

Electron transfer patterns of the di-heme protein cytochrome c4 from Pseudomonas stutzeri

We report kinetic data for the two-step electron transfer (ET) oxidation and reduction of the two-domain di-heme redox protein Pseudomonas stutzeri cytochrome (cyt) c₄ by [Co(bipy)₃]^2+/3+ (bipy = 2,2′-bipyridine). Following earlier reports, the data accord with both bi- and tri-exponential kinetics. A complete kinetic scheme includes both “cooperative” intermolecular ET between each heme group and the external reaction partner, and intramolecular ET between the two heme groups. A new data analysis scheme shows unequivocally that two-ET oxidation and reduction of P. stutzeri cyt c₄ is entirely dominated by intermolecular ET between the heme groups and the external reaction partner in the ms time range, with virtually no contribution from intramolecular interheme ET in this time range. This is in striking contrast to two-ET electrochemical oxidation or reduction of P. stutzeri cyt c₄ for which fast, ms to sub-ms intramolecular interheme ET is a crucial step. The rate constant dependence on the solvent viscosity has disclosed strong coupling to both a (set of) frictionally damped solvent/protein nuclear modes and intramolecular friction-less “ballistic” modes, indicative of notable protein structural mobility in the overall two-ET process. We suggest that conformational protein mobility blocks intramolecular interheme ET in bulk homogeneous solution but triggers opening of this gated ET channel in the electrochemical environment or in the membrane environment of natural respiratory cyt c₄ function.     

The role of a disulfide bridge in the stability and folding kinetics of Arabidopsis thaliana cytochrome c6A

Cytochrome c_6A is a eukaryotic member of the Class I cytochrome c family possessing a high structural homology with photosynthetic cytochrome c₆ from cyanobacteria, but structurally and functionally distinct through the presence of a disulfide bond and a heme mid-point redox potential of + 71 mV (vs normal hydrogen electrode). The disulfide bond is part of a loop insertion peptide that forms a cap-like structure on top of the core α-helical fold. We have investigated the contribution of the disulfide bond to thermodynamic stability and (un)folding kinetics in cytochrome c_6A from Arabidopsis thaliana by making comparison with a photosynthetic cytochrome c₆ from Phormidium laminosum and through a mutant in which the Cys residues have been replaced with Ser residues (C67/73S). We find that the disulfide bond makes a significant contribution to overall stability in both the ferric and ferrous heme states. Both cytochromes c_6A and c₆ fold rapidly at neutral pH through an on-pathway intermediate. The unfolding rate for the C67/73S variant is significantly increased indicating that the formation of this region occurs late in the folding pathway. We conclude that the disulfide bridge in cytochrome c_6A acts as a conformational restraint in both the folding intermediate and native state of the protein and that it likely serves a structural rather than a previously proposed catalytic role.     

Crystal structure of a glutamate/aspartate binding protein complexed with a glutamate molecule: structural basis of ligand specificity at atomic resolution

The crystal structure of a periplasmic l-aspartate/l-glutamate binding protein (DEBP) from Shigella flexneri complexed with an l-glutamate molecule has been determined and refined to an atomic resolution of 1.0 Å. There are two DEBP molecules in the asymmetric unit. The refined model contains 4462 non-hydrogen protein atoms, 730 water molecules, 2 bound glutamate molecules, and 2 Tris molecules from the buffer used in crystallization. The final R_cryst and R_free factors are 13.61% and 16.89%, respectively. The structure has root-mean-square deviations of 0.016 Å from standard bond lengths and 2.35° from standard bond angles.The DEBP molecule is composed of two similarly folded domains separated by the ligand binding region. Both domains contain a central five-stranded β-sheet that is surrounded by several α-helices. The two domains are linked by two antiparallel β-strands. The overall shape of DEBP is that of an ellipsoid approximately 55 Å × 45 Å × 40 Å in size.The binding of ligand to DEBP is achieved mostly through hydrogen bonds between the glutamate and side-chain and main-chain groups of DEBP. Side chains of residues Arg24, Ser72, Arg75, Ser90, and His164 anchor the deprotonated γ-carboxylate group of the glutamate with six hydrogen bonds. Side chains of Arg75 and Arg90 form salt bridges with the deprotonated α-carboxylate group, while the main-chain amide groups of Thr92 and Thr140 form hydrogen bonds with the same group. The positively charged α-amino group of the l-glutamate forms salt bridge interaction with the side-chain carboxylate group of Asp182 and hydrogen bond interaction with main-chain carbonyl oxygen of Ser90. In addition to these hydrogen bond and electrostatic interactions, other interactions may also play important roles. For example, the two methylene groups from the glutamate form van der Waals interactions with hydrophobic side chains of DEBP.Comparisons with several other periplasmic amino acid binding proteins indicate that DEBP residues involved in the binding of α-amino and α-carboxylate groups of the ligand and the pattern of hydrogen bond formation between these groups are very well conserved, but the binding pocket around the ligand side chain is not, leading to the specificity of DEBP. We have identified structural features of DEBP that determine its ability of binding glutamate and aspartate, two molecules with different sizes, but discriminating against very similar glutamine and asparagine molecules.     

Accommodation of NO in the active site of mammalian and bacterial cytochrome c oxidase aa3

Following different reports on the stoichiometry and configuration of NO binding to mammalian and bacterial reduced cytochrome c oxidase aa₃ (CcO), we investigated NO binding and dynamics in the active site of beef heart CcO as a function of NO concentration, using ultrafast transient absorption and EPR spectroscopy. We find that in the physiological range only one NO molecule binds to heme a₃, and time-resolved experiments indicate that even transient binding to Cu_B does not occur. Only at very high (∼ 2 mM) concentrations a second NO is accommodated in the active site, although in a different configuration than previously observed for CcO from Paracoccus denitrificans [E. Pilet, W. Nitschke, F. Rappaport, T. Soulimane, J.-C. Lambry, U. Liebl and M.H. Vos. Biochemistry 43 (2004) 14118-14127], where we proposed that a second NO does bind to Cu_B. In addition, in the bacterial enzyme two NO molecules can bind already at NO concentrations of ∼ 1 μM. The unexpected differences highlighted in this study may relate to differences in the physiological relevance of the CcO-NO interactions in both species.     

Synthesis, crystal structure and luminescent behaviour of coordination complexes of copper with bi- and tridentate amines and phosphonic acids

The synthesis and crystal structure of four new copper(I) and copper(II) supramolecular amine, and amine phosphonate, complexes is reported. Reaction of copper(I) with 2-,9-dimethyl-1-10-phenanthroline (dmp) produced a stable 4-coordinate Cu(I) species, [Cu^(I)(dmp)₂]Cl · MeOH · 5H₂O (2), i.e., the increased steric hindrance in the ‘bite’ area of dmp did not prevent interaction with the metal and provided protection against oxidation which was not possible for the phen analogue [R. Clarke, K. Latham, C. Rix, M. Hobday, J. White, CrystEngCommun. 7(3) (2005), 28-36]. Subsequent addition of phenylphosphonic acid to (2) produced two structures from alternative synthetic routes. An ‘in situ’ process yielded red block Cu(I) crystals, [Cu^(I)(dmp)₂] · [C₆H₅PO₃H₂ · C₆H₅PO₃H] (4), whilst recrystallisation of (2) prior to addition of the acid (‘stepwise’ process) produced a green, needle-like Cu(II) complex, [Cu^(II)(dmp) · (H₂O)₂ · C₆H₅PO₂(OH)] [C₆H₅PO₂(OH)] (3). However, addition of excess dmp during the ‘stepwise’ process forced the equilibrium towards product (4) and resulted in an optimum yield (99%). The structure of (4) was similar to the phen analogue, [Cu^(II)Cl(phen)₂] · [C₆H₅PO₂(OH) · C₆H₅PO(OH)₂] (1) [R. Clarke, K. Latham, C. Rix, M. Hobday, J. White, CrystEngCommun. 7(3) (2005), 28-36], but the presence of dmp exerted some influence on global packing, whilst (3) exists as a polymeric layered material. In contrast, reaction of copper(I) with di-2-pyridyl ketone (dpk), followed by phenylphosphonic acid produced purple/blue Cu(II) species, [Cu^(II)(dpk · H₂O)₂] Cl₂ · 4H₂O (5), and [Cu^(II)(dpk · H₂O)₂] · [C₆H₅PO₂(OH)₂ · C₆H₅PO(OH)₂] (6), respectively, i.e., in both cases oxidation of copper occurred. Solid-state luminescence was observed in (2) and (4). The latter showing a 5-fold enhancement in intensity.     

Electron transfer kinetics between soluble modules of Paracoccus denitrificans cytochrome c1 and its physiological redox partners

The transient electron transfer (ET) interactions between cytochrome c₁ of the bc₁-complex from Paracoccus denitrificans and its physiological redox partners cytochrome c₅₅₂ and cytochrome c₅₅₀ have been characterized functionally by stopped-flow spectroscopy. Two different soluble fragments of cytochrome c₁ were generated and used together with a soluble cytochrome c₅₅₂ module as a model system for interprotein ET reactions. Both c₁ fragments lack the membrane anchor; the c₁ core fragment (c_1CF) consists of only the hydrophilic heme-carrying domain, whereas the c₁ acidic fragment (c_1AF) additionally contains the acidic domain unique to P. denitrificans. In order to determine the ionic strength dependencies of the ET rate constants, an optimized stopped-flow protocol was developed to overcome problems of spectral overlap, heme autoxidation and the prevalent non-pseudo first order conditions. Cytochrome c₁ reveals fast bimolecular rate constants (10⁷ to 10⁸ M^− 1 s^− 1) for the ET reaction with its physiological substrates c₅₅₂ and c₅₅₀, thus approaching the limit of a diffusion-controlled process, with 2 to 3 effective charges of opposite sign contributing to these interactions. No direct involvement of the N-terminal acidic c₁-domain in electrostatically attracting its substrates could be detected. However, a slight preference for cytochrome c₅₅₀ over c₅₅₂ reacting with cyochrome c₁ was found and attributed to the different functions of both cytochromes in the respiratory chain of P. denitrificans.     

Structural and functional characterization of the Geobacillus copper nitrite reductase: Involvement of the unique N-terminal region in the interprotein electron transfer with its redox partner

The crystal structures of copper-containing nitrite reductase (CuNiR) from the thermophilic Gram-positive bacterium Geobacillus kaustophilus HTA426 and the amino (N)-terminal 68 residue-deleted mutant were determined at resolutions of 1.3 Å and 1.8 Å, respectively. Both structures show a striking resemblance with the overall structure of the well-known CuNiRs composed of two Greek key β-barrel domains; however, a remarkable structural difference was found in the N-terminal region. The unique region has one β-strand and one α-helix extended to the northern surface of the type-1 copper site. The superposition of the Geobacillus CuNiR model on the electron-transfer complex structure of CuNiR with the redox partner cytochrome c₅₅₁ in other denitrifier system led us to infer that this region contributes to the transient binding with the partner protein during the interprotein electron transfer reaction in the Geobacillus system. Furthermore, electron-transfer kinetics experiments using N-terminal residue-deleted mutant and the redox partner, Geobacillus cytochrome c₅₅₁, were carried out. These structural and kinetics studies demonstrate that the region is directly involved in the specific partner recognition.