Rhodobacter capsulatus contains a novel cb-type cytochrome c oxidase without a CuA center

The facultative phototrophic bacterium Rhodobacter capsulatus is capable of growth in a wide range of environmental conditions using a highly branched electron-transfer chain. During respiratory growth of this organism reducing equivalents are conveyed to oxygen via two terminal oxidases, previously called "cyt b410" (cytochrome c oxidase) and "cyt b260" (quinol oxidase). The cytochrome c oxidase was purified to homogeneity from a semiaerobically grown R. capsulatus strain. The purified enzyme consumes oxygen at a rate of 600 s-1, oxidizes reduced equine cyt c and R. capsulatus cyt c2, and has high sensitivity to cyanide. The complex is composed of three major polypeptides of apparent molecular masses 45, 32, and 28 kDa on SDS-PAGE. The 32- and 28-kDa proteins also stain with tetramethylbenzidine, indicating that they are c-type cytochromes. Partial amino acid sequences obtained from each of the subunits reveal significant homology to the fixN, fixO, and fixP gene products of Bradyrhizobium japonicum and Rhizobium meliloti. The reduced enzyme has an optical absorption spectrum with distinct features near 550 and 560 nm and an asymmetric Soret band centered at 418 nm, indicating the presence of both c- and b-type cytochromes. Two electrochemically distinct cyt c are apparent, with redox midpoint potentials (Em7) of 265 and 320 mV, while the low-spin cyt b has an Em7 value of 385 mV. The enzyme binds carbon monoxide, and the CO difference spectrum indicates that CO binds to a high-spin cyt b. Pyridine hemochrome and HPLC analyses suggest that the complex contains 1 mol of heme C to 1 mol of protoheme and that neither heme O nor heme A is present.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-molecular-mass multi-c-heme cytochromes from Methylococcus capsulatus bath

The polypeptide and structural gene for a high-molecular-mass c-type cytochrome, cytochrome c553O, was isolated from the methanotroph Methylococcus capsulatus Bath. Cytochrome c553O is a homodimer with a subunit molecular mass of 124,350 Da and an isoelectric point of 6. 0. The heme c concentration was estimated to be 8.2 +/- 0.4 mol of heme c per subunit. The electron paramagnetic resonance spectrum showed the presence of multiple low spin, S = 1/2, hemes. A degenerate oligonucleotide probe synthesized based on the N-terminal amino acid sequence of cytochrome c553O was used to identify a DNA fragment from M. capsulatus Bath that contains occ, the gene encoding cytochrome c553O. occ is part of a gene cluster which contains three other open reading frames (ORFs). ORF1 encodes a putative periplasmic c-type cytochrome with a molecular mass of 118, 620 Da that shows approximately 40% amino acid sequence identity with occ and contains nine c-heme-binding motifs. ORF3 encodes a putative periplasmic c-type cytochrome with a molecular mass of 94, 000 Da and contains seven c-heme-binding motifs but shows no sequence homology to occ or ORF1. ORF4 encodes a putative 11,100-Da protein. The four ORFs have no apparent similarity to any proteins in the GenBank database. The subunit molecular masses, arrangement and number of hemes, and amino acid sequences demonstrate that cytochrome c553O and the gene products of ORF1 and ORF3 constitute a new class of c-type cytochrome.     

Isolation and characterization of nitric oxide reductase from Paracoccus halodenitrificans

Nitric oxide reductase was isolated from the membrane fraction of a denitrifying bacterium, Paracoccus halodenitrificans, in the presence of n-dodecyl beta-D-maltoside. A relatively simple and effective procedure to purify NO reductase using DEAE-Toyopearl and hydroxyapatite (ceramic) chromatographies has been developed. The enzyme consisted of two subunits with molecular masses of 20 and 42 kDa associated with the c-type heme and two b-type hemes, respectively. The optical and magnetic circular dichroism (MCD) spectra of the oxidized (as isolated) and reduced enzymes indicated that the heme c is in the low-spin state and the hemes b are in the high- and low-spin states. The EPR spectrum also showed the presence of the split high-spin component (g perpendicular = 6.6, 6.0) and two low spin components (gz,y,x = 2.96, 2.26, 1.46, gz = 3.59). Although the presence of an extra iron was suggested from atomic absorption spectroscopy, a non-heme iron could not be detected by colorimetric titrations using ferene and 2-(5-nitro-2-pyridylazo)- 5-(N-propyl-N-sulfopropylamino)phenolate (PAPS). One of the extra signals at g = 4.3 and 2.00 might come from a non-heme iron, while they may originate from an adventitious iron and a certain nonmetallic radical, respectively. When CO acted on the reduced enzyme, both of the low-spin hemes were not affected, and when NO acted on the reduced enzyme, the optical and MCD spectra were of a mixture of the oxidized and reduced enzymes. Consequently, the reduction of NO was supposed to take place at the high-spin heme b. The heme c and the low-spin heme b centers were considered to function as electron mediators during the intermolecular and intramolecular processes.     

Intramolecular electron transfer in cytochrome o of Escherichia coli: events following the photolysis of fully and partially reduced CO-bound forms of the bo3 and oo3 enzymes

The events which follow photolysis of CO-inhibited fully reduced and CO-bound mixed-valence cytochrome o have been studied in two variants of the enzyme, one of which contains heme B at the low-spin site (bo3) and the other of which contains heme O (oo3). For this, isolated enzyme was prepared from three different strains of Escherichia coli which produce these two variants in different relative amounts [Puustinen, A., Morgan, J. E., Verkhovsky, M., Thomas, J. W., Gennis, R. B., & Wikstr?m, M. (1992) Biochemistry 31, 10363-10369]. In both types of enzyme microsecond electron redistribution was observed from the oxygen-binding heme to the low-spin heme. In the bo3 enzyme, the rate was similar to that in the bovine enzyme (3 microseconds), but in the oo3 enzyme, it was several times slower. However, in both types of cytochrome o, the same electron redistribution process was also apparently observed on other time scales, some faster and some slower. The rate of CO rebinding in the mixed-valence enzyme was found to be slower than in the fully reduced enzyme, apparently because of the subpopulation of oxidized oxygen-binding heme produced by the electron redistribution. The extent of this electron redistribution, and thus the inter-heme delta Em, can be calculated from this change in rate. The heme B and heme O containing low-spin sites have Em values about 20 and 50 mV lower, respectively, than the oxygen-binding heme.     

Axial coordination and reduction potentials of the sixteen hemes in high-molecular-mass cytochrome c from Desulfovibrio vulgaris (Hildenborough)

A spectroelectrochemical study is described of the sixteen hemes in the high-molecular-mass, monomeric cytochrome c (Hmc) from the periplasmic space of Desulfovibrio vulgaris, strain Hildenborough. One of the hemes has special properties. In the oxidized state at pH 7 it is predominantly high-spin, S = 5/2, with a g perpendicular value of less than 6 indicative of quantum-mechanical mixing with a low-lying (800 cm-1) S = 3/2 state; the balance is probably a low-spin derivative. The high-spin heme has an Em.7.5 value of +61 mV. The fifteen other hemes are low-spin bis-histidine coordinated with Em.7.5 values of approximately -0.20 V. Two of these hemes exhibit very anisotropic EPR spectra with a g1 value of 3.65 characteristic for strained bis-histidine coordination. A previous proposal, namely that methionine is coordinated to one of the hemes [Pollock, W.B.R., Loufti, M. Bruschi, M. Rapp-Giles, B.J., Wall, J. & Voordouw, G. (1991) J. Bacteriol. 173, 220] is disproved using spectroscopic evidence. Contrasting electrochemical data sets from two previous studies [Tan, J. & Cowan, J.A. (1990) Biochemistry 29, 4886; Bruschi, M., Bertrand, P., More, C., Leroy, G., Bonicel, J., Haladjian, J., Chottard, G., Pollock, W.B.R. & Voordouw, G. (1992) Biochemistry 31, 3281] are not consistent with our EPR titration results and are not reproducible. Hmc can be reduced by D. vulgaris Fe-hydrogenase in the presence of molecular hydrogen.     

An engineered cation site in cytochrome c peroxidase alters the reactivity of the redox active tryptophan

The crystal structures of cytochrome c peroxidase and ascorbate peroxidase are very similar, including the active site architecture. Both peroxidases have a tryptophan residue, designated the proximal Trp, located directly adjacent to the proximal histidine heme ligand. During the catalytic cycle, the proximal Trp in cytochrome c peroxidase is oxidized to a cation radical. However, in ascorbate peroxidase, the porphyrin is oxidized, not the proximal Trp, despite the close similarity between the two peroxidase active site structures. A cation located approximately 8 A from the proximal Trp in ascorbate peroxidase but absent in cytochrome c peroxidase is thought to be one reason why ascorbate peroxidase does not form a Trp radical. Site-directed mutagenesis has been used to introduce the ascorbate peroxidase cation binding site into cytochrome c peroxidase. Crystal structures show that mutants now bind a cation. Electron paramagnetic resonance spectroscopy shows that the cation-containing mutants of cytochrome c peroxidase no longer form a stable Trp radical. The activity of the cation mutants using ferrocytochrome c as a substrate is < 1% of wild type levels, while the activity toward a small molecule substrate, guaiacol, increases. These results demonstrate that long range electrostatic effects can control the reactivity of a redox active amino acid side chain and that oxidation/reduction of the proximal Trp is important in the oxidation of ferrocytochrome c.     

Infectious disease complications of simultaneous pancreas kidney transplantation

Gram-positive thermophilic Bacillus species contain cytochrome caa3-type cytochrome c oxidase as their main terminal oxidase in the respiratory chain. To identify alternative oxidases, we isolated several mutants from B. stearothermophilus defective in the caa3-type oxidase activity [Sakamoto, J. et al (1996) FEMS Microbiol. Lett. 143, 151-158]. A novel oxidase was isolated from membrane preparations of one of the mutants, K17. The oxidase was composed of two subunits with molecular masses of 56 and 19 kDa, and contained protoheme IX, heme O, heme A, and Cu in a ratio of 1:0.7:0.2:3. CO difference spectra indicate that the high-spin heme is mainly heme O. These results suggest that the enzyme belongs to the heme-copper oxidase family and is a cytochrome b(o/a)3-type oxidase, whose high-spin heme is mainly heme O and partly heme A. The enzyme oxidized cytochrome c-551, which is a membrane-bound lipoprotein of thermophilic Bacillus. The turnover rate of the activity (Vmax = 190 s[-1]) and its affinity for cytochrome c-551 (Km = 0.15 microM) were much higher than those for yeast and equine heart cytochromes c. The oxidase activity was enhanced by the presence of salts and inhibited by sodium cyanide with a Ki value of 19 microM. The enzyme kinetics suggests that cytochrome c-551 is the natural substrate to this oxidase. Furthermore, the oxidase had similarity to cytochrome ba3-type oxidase from Thermus thermophilus in the subunit composition, partial amino acid sequence, and prosthetic groups, and therefore is suggested to belong to a unique subgroup of the heme-copper oxidase family together with the Thermus enzyme and archaeal oxidases such as Sulfolobus SoxABCD.     

Spectroscopic characterization of recombinant pea cytosolic ascorbate peroxidase: similarities and differences with cytochrome c peroxidase

Recombinant pea cytosolic ascorbate peroxidase (APX) has been characterized by resonance Raman (RR) and electronic absorption spectroscopies. The ferric and ferrous forms together with the complexes with fluoride and imidazole have been studied and compared with the corresponding spectra of cytochrome c peroxidase (CCP). Ferric APX at neutral pH is a mixture of 6- and 5-coordinate high-spin and 6-c low-spin hemes, the latter two species being dominant. The results suggest that the low-spin form derives from a water/hydroxo ligand bound to the heme iron and not from a strong internal ligand as observed in CCP at alkaline pH. Two Fe-Im stretching modes are identified, as in CCP, but the RR frequencies confirm a weaker His163-Asp208 hydrogen bond than in CCP, as suggested on the basis of the X-ray structure [Patterson, W. R., and Poulos, T. L. (1995) Biochemistry 34, 4331-4341]. The data show that CCP and APX have markedly different orientations of the vinyl substituents on the heme chromophore resulting from different steric constraints exerted by the protein matrix.     

Optimal NF kappa B mediated transcriptional responses in Jurkat T cells exposed to oxidative stress are dependent on intracellular glutathione and costimulatory signals

The spectral and electrochemical parameters, as well as the orientations of the heme plane with respect to the membrane plane, of the c-type hemes present in membrane fragments from Heliobacillus mobilis were characterised by optical and EPR spectroscopy. Cytochrome C53, was thereby shown to represent at least four and possibly five heme species with the following characteristics: Em = -60 mV +/- 10 mV, g, = 2.92, 60 degrees; Em = +90 mV +/- 10 mV, g, = 2.92, 90 degrees; Em = +120 mV +/- 20 mV, g, = 3.03; and Em = +170 mV +/- 20 mV, g, = 3.03. The latter component may correspond to two hemes with redox midpoint potentials of Em = +160 mV +/- 20 mV and Em = +180 mV +/- 20 mV (all Em values at pH 7.0). For the heme species having g, peaks at g approximately 3.03, determination of individual orientations was precluded due to the superposition of several differently oriented hemes. About one copy of each heme was found to be present per photosynthetic reaction centre, with the exception of the +120 mV component for which a stoichiometry of 2 hemes/reaction centre was obtained. The heme proteins were detergent-solubilised and partially purified. Three c-type cytochromes that migrated with apparent molecular masses of 18, 29 and 50 kDa were detected on SDS/PAGE. Optical redox titrations at pH 7.0 showed redox midpoint potentials of +160 mV +/- 10 mV for the 18-kDa cytochrome, and -60 mV +/- 10 mV, with possible contributions around +160 mV, for the 50-kDa cytochrome. A tentative attribution of heme species observed in membranes to the isolated heme proteins is presented. The results obtained on H. mobilis are compared with those reported for green sulphur bacteria.     

Spectral and cyanide binding properties of the cytochrome aa3 (600 nm) complex from Bacillus subtilis

The cytochrome aa3 (600 nm) complex, or menaquinol oxidase, from Bacillus subtilis is a member of the cytochrome oxidase superfamily of respiratory membrane protein complexes. We have characterized some spectral properties of this enzyme and its reaction with cyanide. The magnetic circular dichroism (MCD) spectrum of the oxidized enzyme has a single band at 1560 nm in the near-infrared region assigned to bis-histidine-ligated, low-spin ferricytochrome a. The other heme, cytochrome a3, is presumably high-spin in the oxidized enzyme, as isolated. The absence of a trough in the MCD spectrum at 790 nm, observed previously with mammalian cytochrome c oxidase and assigned to CuA (Greenwood et al., Biochem. J. 215, 303-316, 1983), is consistent with the absence of this center from the menaquinol oxidase. When the heme ligand cyanide is added to oxidized menaquinol oxidase, a new MCD band appears at 2010 nm, while the band at 1560 nm is unperturbed. The new band is assigned to low-spin ferricytochrome a3 bound with cyanide. The long-wavelength position of this cyanide-induced band is proposed to arise from the close interaction of cytochrome a3 with the copper atom, CuB. The kinetics of cyanide binding to oxidized cytochrome aa3(600 nm) reveal a spectrally simple, yet kinetically complex process. The reaction is biphasic with second-order rate constants of 45 and 0.61 M-1s-1 at 1 mM KCN, with each phase constituting about 50% of the overall reaction. When the enzyme is subjected to a cycle of anaerobic reduction and air oxidation, the subsequent reaction with cyanide occurs in a single phase at the faster rate. This behavior is ascribed to different conformations of the binuclear center exhibiting different reactivities with cyanide, and is in keeping with that previously established for the structurally more complex mitochondrial cytochrome c oxidase. However, the electronic spectral characteristics of some of the species involved in these reactions are different in the present bacterial case from those of reported eukaryotic systems.     

Recombinant human erythrocyte cytochrome b5

The gene encoding the human erythrocyte form of cytochrome b5 (97 residues in length) has been prepared by mutagenesis of an expression vector encoding lipase-solubilized bovine liver microsomal cytochrome b5 (93 residues in length) (Funk et al., 1990). Efficient expression of this gene in Escherichia coli has provided the first opportunity to obtain this protein in quantities sufficient for physical and functional characterization. Comparison of the erythrocytic cytochrome with the trypsin-solubilized bovine liver cytochrome b5 by potentiometric titration indicates that the principal electrostatic difference between the two proteins results from two additional His residues present in the human erythrocytic protein. The midpoint reduction potential of this protein determined by direct electrochemistry is -9 +/- 2 mV vs SHE at pH 7.0 (mu = 0.10 M, 25.0 degrees C), and this value varies with pH in a fashion that is consistent with the presence of a single ionizable group that changes pKa from 6.0 +/- 0.1 in the ferricytochrome to 6.3 +/- 0.1 in the ferrocytochrome with delta H degrees = -3.2 +/- 0.1 kcal/mol and delta S degrees = -11.5 +/- 0.3 eu (pH 7.0, mu = 0.10). The 1D 1H NMR spectrum of the erythrocytic ferricytochrome indicates that 90% of the protein binds heme in the "major" orientation and 10% of the protein binds heme in the "minor" orientation (pH 7.0, 25 degrees C) with delta H degrees = -2.9 +/- 0.3 kcal/mol and delta S degrees = -5.4 +/- 0.9 eu for this equilibrium.     

Influence of conserved amino acids on the structure and environment of the heme of cytochrome c2. A resonance Raman study

Resonance Raman spectra using Soret excitations of oxidized and reduced Rhodobacter capsulatus cytochrome c2 at pH 7.5 were studied. The spectra of oxidized cytochrome c2 show three components for the v10 mode at 1638, 1633, and 1629 cm(-1). The intensities of these components are sensitive to the excitation wavelength. This effect is explained in the context of a conformational equilibrium of the ferriheme between a nearly planar structure and two ruffled structures. In the case of reduced cytochrome c2, the absolute frequencies as well as the excitation-dependent frequency dispersion of the v10 mode (1618-1621 cm(-1)) indicate a displacement of the conformational equilibrium of heme toward the more planar structures. To measure the influence of some key amino acid residues on the heme-protein interaction of cytochrome c2, four site-directed mutants of Rb. capsulatus cytochrome c2 have been studied by resonance Raman spectroscopy and their spectra compared with the spectra obtained for the wild type cytochrome. The mutants studied are K14E/K32E, P35A, W67Y, and Y75F. The spectral changes induced by the mutations are interpreted in terms of alterations in the structure and/or environment of the cytochrome c2 heme in the framework of the expected role of the different amino acid residues in the stability and redox potential.     

Fast cytochrome bo from Escherichia coli binds two molecules of nitric oxide at CuB

The reaction of nitric oxide (NO) with fast cytochrome bo from Escherichia coli has been studied by electronic absorption, MCD, and EPR spectroscopy. Titration of the enzyme with NO showed the formation of two distinct species, consistent with NO binding stoichiometries of 1:1 and 2:1 with observed dissociation constants at pH 7.5 of approximately 2.3 x 10(-)6 and 3.3 x 10(-)5 M. Monitoring the titration by EPR spectroscopy revealed that the broad EPR signals at g approximately 7.3, 3.7, and 2.8 due to magnetic interaction between high-spin heme o (S = 5/2) and CuBII (S = 1/2) are lost. A high-spin heme o signal at g = 6.0 appears as the 1:1 complex is formed but is lost again on formation of the 2:1 complex, which is EPR silent. The absorption spectrum shows that heme o remains in the high-spin FeIII state throughout the titration. These results are consistent with the binding of up to two NO molecules at CuBII. This has been confirmed by studies with the Cl- adduct of fast cytochrome bo. MCD evidence shows that heme o remains ligated by histidine and water. Addition of excess NO to the Cl- adduct leads to the appearance of a high-spin FeIII heme EPR signal. Hence chloride ion binds to CuB, blocking the binding of a second NO molecule. These results suggest a mechanism for the reduction of NO to nitrous oxide by cytochrome bo and cytochrome c oxidase in which the binding of two cis NO molecules at CuB permits the formation of an N-N bond and the abstraction of oxygen by the heme group.     

A central role for water in the control of the spin state of cytochrome P-450scc

A previous thermodynamic study [Lange, R., Larroque, C. & Anzenbacher, P. (1992) Eur. J. Biochem. 207, 69-73] demonstrated two conformations (A and B) of cytochrome P-450scc (SCC), the enzyme which initiates steroid biosynthesis by cleaving the side chain of cholesterol. The conformation found at the lowest temperatures (form A) displays a six-ligand high-spin heme iron [Hildebrandt, P., Heibel, G., Anzenbacher, P., Lange, R., Krüger, V. & Stier, A. (1994) Biochemistry 33, 12920-12929]. Analytical centrifugation shows that the oligomeric composition of SCC is the same for the A and the B conformers. However, as revealed by fourth-derivative ultraviolet spectroscopy, the two conformers differ in the mean environment of the tryptophan residues, which was more polar in the A form. The structural role of water in these two conformations was investigated using the pressure-jump technique under various pH, temperature and osmotic-stress conditions. Applying hydrostatic pressure to SCC induced very slow (tau >30 min) biexponential relaxation kinetics corresponding to the high-spin to low-spin transition. Analysis of the activation volumes suggested a dissociative mechanism for the A conformer (+45 ml/mol), and an associative mechanism for the B conformer (-39 ml/mol). Applying osmotic stress to the A form changed its kinetic characteristics to those of the B form. These results are consistent with a model comprising a solvent intake (ten water molecules) between the B and the A conformers and protonation of their respective high-spin states. The sixth ligand of the high-spin form in the A conformer involves a water molecule and an unknown constraining structure.     

Purification and characterization of cytochrome c6 from the unicellular green alga Scenedesmus obliquus

Purification of a soluble cytochrome c6 from the unicellular green alga Scenedesmus obliquus by a simple and rapid method is described. The purification procedure includes ammonium sulfate precipitation and non-denaturating PAGE. The N-terminal sequence of the first 20 amino acids was determined and shows 85% similarity and 75% identity to the sequence of cytochrome c6 from the green alga Monoraphidium braunii. The ferrocytochrome shows typical UV/VIS absorption peaks at 552.9, 521.9 and 415.7 nm. The apparent molecular mass was estimated to be 12 kDa by SDS-PAGE. EPR-spectroscopy at 20 K shows resonances indicative for two distinct low-spin heme forms.     

Cytochrome P460 genes from the methanotroph Methylococcus capsulatus bath

P460 cytochromes catalyze the oxidation of hydroxylamine to nitrite. They have been isolated from the ammonia-oxidizing bacterium Nitrosomonas europaea (R. H. Erickson and A. B. Hooper, Biochim. Biophys. Acta 275:231-244, 1972) and the methane-oxidizing bacterium Methylococcus capsulatus Bath (J. A. Zahn et al., J. Bacteriol. 176:5879-5887, 1994). A degenerate oligonucleotide probe was synthesized based on the N-terminal amino acid sequence of cytochrome P460 and used to identify a DNA fragment from M. capsulatus Bath that contains cyp, the gene encoding cytochrome P460. cyp is part of a gene cluster that contains three open reading frames (ORFs), the first predicted to encode a 59,000-Da membrane-bound polypeptide, the second predicted to encode a 12, 000-Da periplasmic protein, and the third (cyp) encoding cytochrome P460. The products of the first two ORFs have no apparent similarity to any proteins in the GenBank database. The overall sequence similarity of the P460 cytochromes from M. capsulatus Bath and N. europaea was low (24.3% of residues identical), although short regions of conserved residues are present in the two proteins. Both cytochromes have a C-terminal, c-heme binding motif (CXXCH) and a conserved lysine residue (K61) that may provide an additional covalent cross-link to the heme (D. M. Arciero and A. B. Hooper, FEBS Lett. 410:457-460, 1997). Gene probing using cyp indicated that a cytochrome P460 similar to that from M. capsulatus Bath may be present in the type II methanotrophs Methylosinus trichosporium OB3b and Methylocystis parvus OBBP but not in the type I methanotrophs Methylobacter marinus A45, Methylomicrobium albus BG8, and Methylomonas sp. strains MN and MM2. Immunoblot analysis with antibodies against cytochrome P460 from M. capsulatus Bath indicated that the expression level of cytochrome P460 was not affected either by expression of the two different methane monooxygenases or by addition of ammonia to the culture medium.     

Dynamics of protein-bound water in the heme domain of P450BM3 studied by high-pressure spectroscopy: comparison with P450cam and P450 2B4

Pressure-induced transitions in the heme domain of cytochrome P450BM3 (P450BMP) were studied versus the concentration of palmitic acid. An increase in hydrostatic pressure causes a high- to low-spin shift and subsequent P450 to P420 transition. Conversion of P450BMP to P420 is associated with important conformational and hydration changes of the protein. Treating the pressure-induced changes in the high-spin content in P450 in terms of the four-state model of spin transitions and substrate binding, we evaluated and compared the barotropic parameters of these transitions for P450MBP, P450cam, and P450 2B4 (2B4). In the current study, the pressure-induced transitions in P450cam were reinvestigated versus the concentration of camphor. The interactions of 2B4 and P450BMP with their substrates (benzphetamine and palmitic acid) were accompanied by larger changes in the partial volume of the proteins (+267 and +248 mL/mol, respectively) than the interactions of P450cam with camphor (+106 mL/mol). For 2B4 and P450BMP, substrate binding apparently requires hydration of regions outside the active site. The reaction volumes of the low- to high-spin transitions of the substrate-free cytochromes (20-23 mL/mol) are consistent with the displacement of one water molecule. The volume changes in the high- to low-spin transition of the substrate-bound P450cam, 2B4, and P450BMP (-90, -49, and -16 mL/mol correspondingly) reveal a linear relationship with DeltaG degrees of the spin transition, suggesting that modulation of the spin state by substrate binding is driven by a common mechanism in all three heme proteins.     

Crystal structure of the di-haem cytochrome c peroxidase from Pseudomonas aeruginosa

BACKGROUND: Cytochrome c peroxidase from Pseudomonas aeruginosa (PsCCP) represents a new class of peroxidases which work without the need to create a semi-stable free radical for catalysis. The enzyme is located in the bacterial periplasm where its likely function is to provide protection against toxic peroxides. The soluble 323-residue single polypeptide chain contains two covalent c-type haems with very different properties: one of them is a low-potential (-330 mV) centre where hydrogen peroxide is reduced (the peroxidatic site); the other is a high-potential (+320 mV) centre which feeds electrons to the peroxidatic site from soluble electron-shuttle proteins such as cytochrome c and azurin. RESULTS: The crystal structure of the oxidized form of PsCCP has been determined to 2.4 A resolution by multiple isomorphous replacement, and refined to an R-factor of 19.2%. PsCCP is organized into two domains, both of them containing a covalent c-haem in a structure reminiscent of class 1 cytochromes c. The domains are related by a quasi-twofold axis. The domain interface holds a newly discovered calcium-binding site with an unusual set of ligands. CONCLUSIONS: The likely function of the calcium site is to maintain the structural integrity of the enzyme and/or to modulate electron transfer between the two haem domains. The low-potential haem has two histidine axial ligands (His55 and His71) and the high-potential haem is ligated by His201 and Met275. There are no polar residues at the peroxidatic site in the inactive oxidized enzyme. The structure suggests that, in the half-reduced functional form of the enzyme, the low-potential haem has to shed His71 in order to make the enzyme catalytically competent. This process is likely to trigger a reorganization of the active site, and may introduce a new residues into the haem pocket.     

Altering substrate specificity at the heme edge of cytochrome c peroxidase

Two mutants of cytochrome c peroxidase (CCP) are reported which exhibit unique specificities toward oxidation of small substrates. Ala-147 in CCP is located near the delta-meso edge of the heme and along the solvent access channel through which H2O2 is thought to approach the active site. This residue was replaced with Met and Tyr to investigate the hypothesis that small molecule substrates are oxidized at the exposed delta-meso edge of the heme. X-ray crystallographic analyses confirm that the side chains of A147M and A147Y are positioned over the delta-meso heme position and might therefore modify small molecule access to the oxidized heme cofactor. Steady-state kinetic measurements show that cytochrome c oxidation is enhanced 3-fold for A147Y relative to wild type, while small molecule oxidation is altered to varying degrees depending on the substrate and mutant. For example, oxidation of phenols by A147Y is reduced to less than 20% relative to the wild-type enzyme, while Vmax/e for oxidation of other small molecules is less affected by either mutation. However, the "specificity" of aniline oxidation by A147M, i.e., (Vmax/e)/Km, is 43-fold higher than in wild-type enzyme, suggesting that a specific interaction for aniline has been introduced by the mutation. Stopped-flow kinetic data show that the restricted heme access in A147Y or A147M slows the reaction between the enzyme and H202, but not to an extent that it becomes rate limiting for the oxidation of the substrates examined. The rate constant for compound ES formation with A147Y is 2.5 times slower than wild-type CCP. These observations strongly support the suggestion that small molecule oxidations occur at sites on the enzyme distinct from those utilized by cytochrome c and that the specificity of small molecule oxidation can be significantly modulated by manipulating access to the heme edge. The results help to define the role of alternative electron transfer pathways in cytochrome c peroxidase and may have useful applications in improving the specificity of peroxidase with engineered function.     

Characterization of C415 mutants of neuronal nitric oxide synthase

Nitric oxide synthase (NOS) catalyzes the oxidation of L-arginine to citrulline and nitric oxide. C415H and C415A mutants of the neuronal isoform of NOS (nNOS) were expressed in a baculovirus system and purified to homogeneity for spectral analysis and activity measurements. UV-visible spectra of each mutant lacked an observable Soret peak, suggesting that neither mutant contained heme. When reduced in the presence of CO, however, a small Soret centered at 417 nm could be detected for the C415H mutant, further supporting the assignment of C415 as the axial ligand to the heme. In addition to a deficiency in bound heme, neither mutant had any detectable bound tetrahydrobiopterin, as compared to wild-type enzyme, which had a ratio of 0.84 mol of bound pteridine:1 mol of nNOS 160 kDa subunit. The C415H mutant contained bound FAD and FMN at levels of 1.0 +/- 0.1 and 0.9 +/- 0.1 mol/mol of nNOS subunit, respectively. UV-visible spectra of both nNOS mutants retained the distinctive absorbance due to tightly associated oxidized flavin prosthetic groups. Further, the spectra suggested the presence of a neutral flavin semiquinone. Ferricyanide oxidation of the C415A mutant yielded a spectrum that was essentially that of oxidized flavin. Ferricyanide titration showed that the C415A mutant contained approximately 1 reducing equiv. Circular dichroism spectra suggested that each mutant was folded properly, in that both spectra were found to be essentially identical to the spectrum of wild-type nNOS. Neither mutant could synthesize nitric oxide, and neither mutant had the ability to oxidize NADPH unless an exogenous electron acceptor was added. The rate of cytochrome c reduction by each mutant was found to be slightly less, but very similar to the rate (approximately 20 mumol mg-1 min-1) observed with wild-type nNOS. In all cases, the rate of cytochrome c reduction increased approximately 15-fold with the addition of calmodulin. Overall, these spectral and activity data suggest that C415 is the axial heme ligand and that a point mutation at C415 prevents binding of heme and tetrahydrobiopterin without interfering with the global folding or the reductase function of nNOS.