Cytochrome cd1 nitrite reductase structure raises interesting mechanistic questions.
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Cytochromes↗
Biomedical subjects
S J Ferguson
Publications and source records attributed to S J Ferguson.
The influence of the acetabular labrum on hip joint cartilage consolidation: a poroelastic finite element model.
The goal of this study was to investigate the influence of the acetabular labrum on the consolidation, and hence the solid matrix strains and stresses, of the cartilage layers of the hip joint. A plane-strain finite element model was developed, which represented a coronal slice through the acetabular and femoral cartilage layers and the acetabular labrum. Elements with poroelastic properties were used to account for the biphasic solid/fluid nature of the cartilage and labrum. The response of the joint over an extended period of loading (10,000s) was examined to simulate the nominal compressive load that the joint is subjected to throughout the day. The model demonstrated that the labrum adds an important resistance in the flow path of the fluid being expressed from the cartilage layers of the joint. Cartilage layer consolidation was up to 40% quicker in the absence of the labrum. Following removal of the labrum from the model, the solid-on-solid contact stresses between the femoral and acetabular cartilage layers were greatly increased (up to 92% higher), which would increase the friction between the joint surfaces. In the absence of the labrum, the centre of contact shifted towards the acetabular rim. Subsurface strains and stresses were much higher without the labrum, which could contribute to fatigue damage of the cartilage layers. Finally, the labrum provided some structural resistance to lateral motion of the femoral head within the acetabulum, enhancing joint stability and preserving joint congruity.
Acetabulum↗
The acetabular labrum seal: a poroelastic finite element model.
OBJECTIVE: The aim of the study is to investigate the labrum's ability to seal a pressurised layer of synovial fluid within the joint, and to study the influence of this sealing mechanism on cartilage deformation, interstitial fluid pressure and collagen solid matrix stresses. BACKGROUND: Cartilage degeneration has been observed in conjunction with labrum pathology. However, little is known about the function of the labrum. Experimental observations have been reported which are consistent with a sealing function of the labrum. METHODS: The model was an axisymmetric geometric approximation of the acetabular and femoral cartilage layers and the surrounding labrum. A poroelastic formulation was used to account for the solid and fluid components of these hydrated tissues. A sensitivity analysis of the labrum material properties was carried out. RESULTS: With a compressive load of 1200 N applied across the joint model, the labrum could seal a layer of pressurised fluid between the femur and acetabulum, thus preventing contact of the articulating surfaces. With this sealing effect, loads were transferred across the joint predominantly by uniform pressurisation of the interstitial fluid of the cartilage layers. In the absence of this sealing, strains within the solid matrix of the cartilage layers were higher (e.g. 20% vs. 3%). CONCLUSIONS: The labrum can seal against fluid expression from the joint space. This sealing function protects the cartilage layers of the hip. RELEVANCE: Current treatments for labrum damage and early arthrosis may compromise the sealing function of the labrum. With continued study of the function and importance of the labrum, new surgical repair strategies can be developed to maintain the overall function of the hip joint.
Acetabulum↗
A switch in heme axial ligation prepares Paracoccus pantotrophus cytochrome cd1 for catalysis.
Cytochrome cd1 nitrite reductase (cd1) from Paracoccus pantotrophus is a respiratory enzyme capable of using nitrite, hydroxylamine and oxygen as electron accepting substrates. Structural studies have shown that when the enzyme is reduced there is a change in the axial ligation of both hemes, which has been proposed to form part of the catalytic cycle. Here we report the use of a physiological electron donor, pseudoazurin, to investigate the relationship between heme ligation and catalysis. A combination of visible absorption and electron paramagnetic resonance spectroscopies reveals the formation of a catalytically competent state of oxidized cd1 with 'switched' axial ligands immediately after complete reoxidation of reduced cd1 with hydroxylamine. This activated conformer returns over 20 min at 25 degrees C to the state previously observed for oxidized 'as isolated' cd1, which is catalytically inactive towards the same substrates.
Catalysis↗
Escherichia coli DipZ: anatomy of a transmembrane protein disulphide reductase in which three pairs of cysteine residues, one in each of three domains, contribute differentially to function.
DipZ is a bacterial cytoplasmic membrane protein that transfers reducing power from the cytoplasm to the periplasm so as to facilitate the formation of correct disulphide bonds and c-type cytochromes in the latter compartment. Topological analysis using gene fusions between the Escherichia coli dipZ and either E. coli phoA or lacZ shows that DipZ has a highly hydrophobic central domain comprising eight transmembrane alpha-helices plus periplasmic globular N-terminal and C-terminal domains. The previously assigned translational start codon for the E. coli DipZ was shown to be incorrect and the protein to be larger than previously thought. The experimentally determined translational start position indicates that an additional alpha-helix at the N-terminus acts as a cleavable signal peptide so that the N-terminus of the mature protein is located in the periplasm. The newly assigned 5' end of the dipZ gene was shown to be preceded by a functional ribosome-binding site. The hydrophobic central domain and both of the periplasmic globular domains each have a pair of highly conserved cysteine residues, and it was shown by site directed mutagenesis that all six conserved cysteine residues contribute to DipZ function.
Alkaline Phosphatase↗
Crystallization and preliminary X-ray analysis of nitrous oxide reductase from Paracoccus pantotrophus.
Nitrous oxide reductase is a periplasmic respiratory protein with a novel copper catalytic centre; it catalyses the terminal step, reduction of nitrous oxide to nitrogen, of the bacterial denitrification process. Nitrous oxide reductase from Paracoccus pantotrophus has been crystallized by the hanging-drop method. A prerequisite for crystallization was the oxidation of the enzyme with potassium ferricyanide in order to obtain homogenous oxidation states of the copper centres. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 116.4, b = 118.3, c = 267.0 A. Two homodimers, of approximate molecular weight 67 kDa per subunit, probably constitute the asymmetric unit and give a Matthews coefficient, V(m), of 3.4 A(3) Da(-1) and a solvent content of 59% by volume. The crystals diffract X-rays to 3.0 A resolution on an in-house source and are suitable for structure determination.
Crystallization↗
The structure and dynamics in solution of Cu(I) pseudoazurin from Paracoccus pantotrophus.
The solution structure and backbone dynamics of Cu(I) pseudoazurin, a 123 amino acid electron transfer protein from Paracoccus pantotrophus, have been determined using NMR methods. The structure was calculated to high precision, with a backbone RMS deviation for secondary structure elements of 0.35+/-0.06 A, using 1,498 distance and 55 torsion angle constraints. The protein has a double-wound Greek-key fold with two alpha-helices toward its C-terminus, similar to that of its oxidized counterpart determined by X-ray crystallography. Comparison of the Cu(I) solution structure with the X-ray structure of the Cu(II) protein shows only small differences in the positions of some of the secondary structure elements. Order parameters S2, measured for amide nitrogens, indicate that the backbone of the protein is rigid on the picosecond to nanosecond timescale.
Amides↗
Stabilization of Pseudomonas aeruginosa cytochrome c(551) by systematic amino acid substitutions based on the structure of thermophilic Hydrogenobacter thermophilus cytochrome c(552).
A heterologous overexpression system for mesophilic Pseudomonas aeruginosa holocytochrome c(551) (PA c(551)) was established using Escherichia coli as a host organism. Amino acid residues were systematically substituted in three regions of PA c(551) with the corresponding residues from thermophilic Hydrogenobacter thermophilus cytochrome c(552) (HT c(552)), which has similar main chain folding to PA c(551), but is more stable to heat. Thermodynamic properties of PA c(551) with one of three single mutations (Phe-7 to Ala, Phe-34 to Tyr, or Val-78 to Ile) showed that these mutants had increased thermostability compared with that of the wild-type. Ala-7 and Ile-78 may contribute to the thermostability by tighter hydrophobic packing, which is indicated by the three dimensional structure comparison of PA c(551) with HT c(552). In the Phe-34 to Tyr mutant, the hydroxyl group of the Tyr residue and the guanidyl base of Arg-47 formed a hydrogen bond, which did not exist between the corresponding residues in HT c(552). We also found that stability of mutant proteins to denaturation by guanidine hydrochloride correlated with that against the thermal denaturation. These results and others described here suggest that significant stabilization of PA c(551) can be achieved through a few amino acid substitutions determined by molecular modeling with reference to the structure of HT c(552). The higher stability of HT c(552) may in part be attributed to some of these substitutions.
Amino Acid Substitution↗
Still a puzzle: why is haem covalently attached in c-type cytochromes?
c-Type cytochromes are a group of proteins with diverse structures and functions. Their common feature is covalent attachment of haem to one or more CXXCH motifs. There does not seem to be a single advantageous reason for this covalent attachment.
Amino Acid Sequence↗
Models for molybdenum coordination during the catalytic cycle of periplasmic nitrate reductase from Paracoccus denitrificans derived from EPR and EXAFS spectroscopy.
The periplasmic nitrate reductase from Paracoccus denitrificans is a soluble two-subunit enzyme which binds two hemes (c-type), a [4Fe-4S] center, and a bis molybdopterin guanine dinucleotide cofactor (bis-MGD). A catalytic cycle for this enzyme is presented based on a study of these redox centers using electron paramagnetic resonance (EPR) and extended X-ray absorption fine structure (EXAFS) spectroscopies. The Mo(V) EPR signal of resting NAP (High g [resting]) has g(av) = 1.9898 is rhombic, exhibits low anisotropy, and is split by two weakly interacting protons which are not solvent-exchangeable. Addition of exogenous ligands to this resting state (e.g., nitrate, nitrite, azide) did not change the form of the signal. A distinct form of the High g Mo(V) signal, which has slightly lower anisotropy and higher rhombicity, was trapped during turnover of nitrate and may represent a catalytically relevant Mo(V) intermediate (High g [nitrate]). Mo K-edge EXAFS analysis was undertaken on the ferricyanide oxidized enzyme, a reduced sample frozen within 10 min of dithionite addition, and a nitrate-reoxidized form of the enzyme. The oxidized enzyme was fitted best as a di-oxo Mo(VI) species with 5 sulfur ligands (4 at 2. 43 A and 1 at 2.82 A), and the reduced form was fitted best as a mono-oxo Mo(IV) species with 3 sulfur ligands at 2.35 A. The addition of nitrate to the reduced enzyme resulted in reoxidation to a di-oxo Mo(VI) species similar to the resting enzyme. Prolonged incubation of NAP with dithionite in the absence of nitrate (i.e., nonturnover conditions) resulted in the formation of a species with a Mo(V) EPR signal that is quite distinct from the High g family and which has a g(av) = 1.973 (Low g [unsplit]). This signal resembles those of the mono-MGD xanthine oxidase family and is proposed to arise from an inactive form of the nitrate reductase in which the Mo(V) form is only coordinated by the dithiolene of one MGD. In samples of NAP that had been reduced with dithionite, treated with azide or cyanide, and then reoxidized with ferricyanide, two Mo(V) signals were detected with g(av) elevated compared to the High g signals. Kinetic analysis demonstrated that azide and cyanide displayed competitive and noncompetitive inhibition, respectively. EXAFS analysis of azide-treated samples show improvement to the fit when two nitrogens are included in the molybdenum coordination sphere at 2.52 A, suggesting that azide binds directly to Mo(IV). Based on these spectroscopic and kinetic data, models for Mo coordination during turnover have been proposed.
Azides↗
The Greek key protein apo-pseudoazurin folds through an obligate on-pathway intermediate.
Folding of the 123 amino acid residue Greek key protein apo-pseudo azurin from Thiosphaera pantotropha has been examined using stopped-flow circular dichroism in 0.5 M Na2SO4 at pH 7.0 and 15 degrees C. The data show that the protein folds from the unfolded state with all eight proline residues in their native isomers (seven trans and one cis) to an intermediate within the dead-time of the stopped-flow mixing (50 ms). The urea dependence of the rates of folding and unfolding of the protein were also determined. The ratio of the folding rate to the unfolding rate (extrapolated into water) is several orders of magnitude too small to account for the equilibrium stability of the protein, consistent with the population of an intermediate. Despite this, the logarithm of the rate of folding versus denaturant concentration is linear. These data can be rationalised by the population of an intermediate under all refolding conditions. Accordingly, kinetic and equilibrium measurements were combined to fit the chevron plot to an on-pathway model (U <==> I <==> N). The fit shows that apo-pseudoazurin rapidly forms a compact species that is stabilised by 25 kJ/mol before folding to the native state at a rate of 2 s-1. Although the data can also be fitted to an off-pathway model (I <==> U <==> N), the resulting kinetic parameters indicate that the protein would have to fold to the native state at a rate of 86,000 s-1 (a time constant of only 12 microseconds). Similarly, models in which this intermediate is bypassed also lead to unreasonably fast refolding rates. Thus, the intermediate populated during the refolding of apo-pseudoazurin appears to be obligate and on the folding pathway. We suggest, based on this study and others, that some intermediates play a critical role in limiting the search to the native state.
Apoproteins↗
Three-dimensional computational reconstruction of mixed anatomical tissues following histological preparation.
The creation of geometrically accurate computer models of anatomical structures with complex shape and mixed tissue types can be difficult. A method for shape reconstruction based on digital images of polymer embedded, serially sectioned specimens is presented. The distortion of bone and soft tissue specimens during all stages of histological preparation was measured. Serial sections of one specimen were stained with common histological stains to enhance the contrast between different tissue types. High-resolution digital images of these sections were then processed into a three-dimensional solid model using commercial software. Preparations containing bone and cartilaginous tissues were dimensionally stable following fixation, dehydration and embedding (shrinkage/expansion less than 2%). Staining was necessary to identify anatomical features that otherwise could not be differentiated from their surroundings. Although time consuming, this method provides cross-section images of a higher resolution than those obtained from CT or MRI scanning, and with better soft tissue visualisation.
Animals↗
Biomechanical analysis of dynamic external fixation devices for the treatment of distal radial fractures.
BACKGROUND: Several dynamic external fixation devices have been introduced to permit early functional treatment of unstable distal radial fractures. METHODS: An intact cadaver wrist was spanned by a dynamic external fixator. Forces between the fixator and the radius were recorded during passive motion using a single, strain-gauged pin. A device with a double ball joint was compared with a new, experimental fixator with 3 degrees of freedom and its center of rotation coincident with that of the wrist. RESULTS: During flexion and extension, the pin load was approximately the same for both fixators. Radioulnar deviation resulted in high pin loads for the fixator with ball joints; the new device showed no considerable pin load. CONCLUSION: The new device has a high degree of kinematic compatibility with the natural wrist joint. When used for the treatment of distal radial fractures, it should provide favorable conditions for the maintenance of fracture reduction compared with a device with an offset ball joint.
Biomechanical Phenomena↗
Spectroscopic characterization of a novel multiheme c-type cytochrome widely implicated in bacterial electron transport.
NapC is a member of a family of bacterial membrane-anchored tetra-heme c-type cytochromes that participate in a number of respiratory electron transport pathways. They are postulated to mediate electron transfer between membrane quinols/quinones and soluble periplasmic enzymes. The water-soluble heme domain of NapC has been expressed as a periplasmic protein. Mediated redox potentiometry and characterization by UV-visible, magnetic circular dichroism, and electron paramagnetic resonance spectroscopies demonstrates that soluble NapC contains four low spin hemes, each with bis-histidine axial ligation and with midpoint reduction potentials of -56, -181, -207, and -235 mV.
Amino Acid Sequence↗
An Escherichia coli ccm (cytochrome c maturation) deletion strain substantially expresses Hydrogenobacter thermophilus cytochrome c552 in the cytoplasm: availability of haem influences cytochrome c552 maturation.
The maturation of Hydrogenobacter thermophilus cytochrome c552 in the cytoplasm of Escherichia coli is unique among bacterial c-type cytochromes. It is now shown to be matured in a strain lacking the whole set of ccm (cytochrome c maturation) genes that are normally required for c-type cytochrome biogenesis in E. coli. As this cytochrome is thermostable we propose that the apocytochrome c552 has sufficient tertiary structure to allow the haem to slot into its binding pocket, which in turn triggers the spontaneous covalent attachment between apocytochrome c552 and haem. The ccm deletion strain of E. coli, derived from a strain that synthesizes elevated levels of endogenous c-type cytochromes, also produces larger amounts of cytoplasmic H. thermophilus cytochrome c552 than a reference strain. This implies that elevated production of c-type cytochromes is not a consequence of high activity of ccm genes but rather an enhanced ability to supply haem, a view that is supported by the increase in thermophilic cytochrome c552 biogenesis that occurs in a reference strain following supplementation of growth media with delta-aminolevulinic acid.
Aminolevulinic Acid↗
Contrasting routes of c-type cytochrome assembly in mitochondria, chloroplasts and bacteria.
The biogenesis of bacterial c-type cytochromes generally involves many gene products--some of which may also have roles in other processes--and their interaction with the disulphide-bond-forming system of the bacterial periplasm. However, in some bacteria a simpler process appears to operate that might be related to the formation of c-type cytochromes in thylakoids of photosynthetic cells. The corresponding process in fungal mitochondria is distinct.
Bacteria↗
Nitrogen cycle enzymology.
The minimal nitrogen cycle involves five reduction reactions and three oxidation reactions, each of which poses interesting problems in bioinorganic chemistry, energy transduction and protein structure/function relationships. Many of the major recent developments in this field have depended on the acquisition of protein crystal structures, including structures of enzymes with bound substrates or products and in protein-protein complexes. These enzymes include nitrogenase, nitrite reductases, hydroxylamine oxidoreductase and a fungal nitric oxide reductase.
Adenosine Triphosphate↗
The diversity of redox proteins involved in bacterial heterotrophic nitrification and aerobic denitrification.
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Aerobiosis↗