Heme A Synthase Deficiency Affects the Ability of Bacillus cereus to Adapt to a Nutrient-Limited Environment.

The branched aerobic respiratory chain in Bacillus cereus comprises three terminal oxidases: cytochromes aa3, caa3, and bd. Cytochrome caa3 requires heme A for activity, which is produced from heme O by heme A synthase (CtaA). In this study, we deleted the ctaA gene in B. cereus AH187 strain, this deletion resulted in loss of cytochrome caa3 activity. Proteomics data indicated that B. cereus grown in glucose-containing medium compensates for the loss of cytochrome caa3 activity by remodeling its respiratory metabolism. This remodeling involves up-regulation of cytochrome aa3 and several proteins involved in redox stress response-to circumvent sub-optimal respiratory metabolism. CtaA deletion changed the surface-composition of B. cereus, affecting its motility, autoaggregation phenotype, and the kinetics of biofilm formation. Strikingly, proteome remodeling made the ctaA mutant more resistant to cold and exogenous oxidative stresses compared to its parent strain. Consequently, we hypothesized that ctaA inactivation could improve B. cereus fitness in a nutrient-limited environment.

Response of Heme Symmetry to the Redox State of Bovine Cytochrome c Oxidase.

Second-derivative absorption spectroscopy was employed to monitor the response of effective symmetry of cytochromes a and a3 to the redox and ligation states of bovine cytochrome c oxidase (CcO). The Soret band π → π* electronic transitions were used to display the changes in symmetry of these chromophores induced by the reduction of CcO inhibited by the exogenous ligands and during catalytic turnover. The second derivative of the difference absorption spectra revealed only a single Soret band for the oxidized cytochromes a and a3 and cyanide-ligated oxidized cytochrome a3. In contrast, two absorption bands were resolved in ferrous cytochrome a and ferrous cytochrome a3 ligated with cyanide. A transition from one-band spectrum to two-band spectrum indicates the lowering of symmetry of these hemes due to the alteration of their immediate surroundings. It is suggested that the changes in polarity occurring in the vicinity of these cofactors are main reason for the split of the Soret band of both ferrous cytochrome a and cyanide-bound ferrous cytochrome a3.

Structure of the alternative complex III in a supercomplex with cytochrome oxidase.

Alternative complex III (ACIII) is a key component of the respiratory and/or photosynthetic electron transport chains of many bacteria1-3. Like complex III (also known as the bc1 complex), ACIII catalyses the oxidation of membrane-bound quinol and the reduction of cytochrome c or an equivalent electron carrier. However, the two complexes have no structural similarity4-7. Although ACIII has eluded structural characterization, several of its subunits are known to be homologous to members of the complex iron-sulfur molybdoenzyme (CISM) superfamily 8 , including the proton pump polysulfide reductase9,10. We isolated the ACIII from Flavobacterium johnsoniae with native lipids using styrene maleic acid copolymer11-14, both as an independent enzyme and as a functional 1:1 supercomplex with an aa3-type cytochrome c oxidase (cyt aa3). We determined the structure of ACIII to 3.4 Å resolution by cryo-electron microscopy and constructed an atomic model for its six subunits. The structure, which contains a [3Fe-4S] cluster, a [4Fe-4S] cluster and six haem c units, shows that ACIII uses known elements from other electron transport complexes arranged in a previously unknown manner. Modelling of the cyt aa3 component of the supercomplex revealed that it is structurally modified to facilitate association with ACIII, illustrating the importance of the supercomplex in this electron transport chain. The structure also resolves two of the subunits of ACIII that are anchored to the lipid bilayer with N-terminal triacylated cysteine residues, an important post-translational modification found in numerous prokaryotic membrane proteins that has not previously been observed structurally in a lipid bilayer.

IN VIVO EFFECT OF RUTA CHALEPENSIS EXTRACT ON HEPATIC CYTOCHROME 3A1 IN RATS.

BACKGROUND: Since the time when drugs began to be used, it became evident that they could produce a therapeutic effect, but also a clinical condition of toxicity or no effect at all on humans, despite using the same doses in different patients. Such untoward effects were termed "drug idiosyncrasy" and also "idiosyncratic drug effects", but the factors producing such diverse responses were never taken into account. MATERIALS AND METHODS: Ruta chalepensis L. (fringed rue) is an herbaceous plant of the Rutaceae family used in traditional medicine due to its properties, such as its analgesic and antipyretic effects. This study used 25 male rats divided into five groups. Plant extract was administered to Groups 1 and 2 at doses of 100 and 30 mg/kg/day, respectively, for three days; Group 3 was administered 100 mg/kg/day of dexamethasone (DEX), as well as 100 mg/kg/day of Ruta chalepensis extract; Group 4 was administered 100 mg/kg/day of DEX and treated as positive control; Group 5 was treated as negative control and was administered a physiological solution. Twenty-four hours after the the last dose, the animals were sacrificed and their livers were extracted. RESULTS: The aqueous extract of Ruta chalepensis, intraperitoneally administered, was able to induce cytochrome 3A1 in doses of 30 mg/kg/day, and a greater inducing effect occurs when the plant is co-administered in doses of 100 mg/kg/day with dexamethasone. CONCLUSION: This study suggests that aqueous extract of Ruta chalepensis can induce cytochrome 3a1. This study helps provide a better understanding of CYP3a regulation. Future in vitro work is needed to determine the compounds that produce the cytochrome modulation.

The monoheme cytochrome c subunit of Alternative Complex III is a direct electron donor to caa3 oxygen reductase in Rhodothermus marinus.

Alternative Complex III (ACIII) is an example of the robustness and flexibility of prokaryotic respiratory chains. It performs quinol:cytochrome c oxidoreductase activity, being functionally equivalent to the bc1 complex but structurally unrelated. In this work we further explored ACIII investigating the role of its monoheme cytochrome c subunit (ActE). We expressed and characterized the individually isolated ActE, which allowed us to suggest that ActE is a lipoprotein and to show its function as a direct electron donor to the caa3 oxygen reductase.

Nitric oxide activation by caa3 oxidoreductase from Thermus thermophilus.

Visible and UV-resonance Raman spectroscopy was employed to investigate the reaction of NO with cytochrome caa3 from Thermus thermophilus. We show the formation of the hyponitrite (HO-N=N-O)(-) bound to the heme a3 species (νN=N = 1330 cm(-1)) forming a high spin complex in the oxidized heme a3 Fe/CuB binuclear center of caa3-oxidoreductase. In the absence of heme a3 Fe(2+)-NO formation, the electron required for the formation of the N=N bond originates from the autoreduction of CuB by NO, producing nitrite. With the identification of the hyponitrite intermediate the hypothesis of a common phylogeny of aerobic respiration and bacterial denitrification is fully supported and the mechanism for the 2e(-)/2H(+) reduction of NO to N2O can be described with more certainty.

Structure of caa(3) cytochrome c oxidase--a nature-made enzyme-substrate complex.

Aerobic respiration, the energetically most favorable metabolic reaction, depends on the action of terminal oxidases that include cytochrome c oxidases. The latter forms a part of the heme-copper oxidase superfamily and consists of three different families (A, B, and C types). The crystal structures of all families have now been determined, allowing a detailed structural comparison from evolutionary and functional perspectives. The A2-type oxidase, exemplified by the Thermus thermophilus caa(3) oxidase, contains the substrate cytochrome c covalently bound to the enzyme complex. In this article, we highlight the various features of caa(3) enzyme and provide a discussion of their importance, including the variations in the proton and electron transfer pathways.

Structural insights into electron transfer in caa3-type cytochrome oxidase.

Cytochrome c oxidase is a member of the haem copper oxidase superfamily (HCO). HCOs function as the terminal enzymes in the respiratory chain of mitochondria and aerobic prokaryotes, coupling molecular oxygen reduction to transmembrane proton pumping. Integral to the enzyme's function is the transfer of electrons from cytochrome c to the oxidase via a transient association of the two proteins. Electron entry and exit are proposed to occur from the same site on cytochrome c. Here we report the crystal structure of the caa3-type cytochrome oxidase from Thermus thermophilus, which has a covalently tethered cytochrome c domain. Crystals were grown in a bicontinuous mesophase using a synthetic short-chain monoacylglycerol as the hosting lipid. From the electron density map, at 2.36 Å resolution, a novel integral membrane subunit and a native glycoglycerophospholipid embedded in the complex were identified. Contrary to previous electron transfer mechanisms observed for soluble cytochrome c, the structure reveals the architecture of the electron transfer complex for the fused cupredoxin/cytochrome c domain, which implicates different sites on cytochrome c for electron entry and exit. Support for an alternative to the classical proton gate characteristic of this HCO class is presented.

Electron transfer dynamics of Rhodothermus marinus caa3 cytochrome c domains on biomimetic films.

The subunit II of the caa(3) oxygen reductase from Rhodothermus marinus contains, in addition to the Cu(A) center, a c-type heme group in the cytochrome c domain (Cyt-D) that is the putative primary electron acceptor of the enzyme. In this work we have combined surface-enhanced resonance Raman (SERR) spectroelectrochemistry, molecular dynamics (MD) simulations and electron pathway calculations to assess the most likely interaction domains and electron entry/exit points of the truncated Cyt-D of subunit II in the reactions with its electron donor, HiPIP and electron acceptor, Cu(A). The results indicate that the transient interaction between Cyt-D and HiPIP relies upon a delicate balance of hydrophobic and polar contacts for establishing an optimized electron transfer pathway that involves the exposed edge of the heme group and guaranties efficient inter-protein electron transfer on the nanosecond time scale. The reorganization energy of ca. 0.7 eV was determined by time-resolved SERR spectroelectrochemistry. The intramolecular electron transfer pathway in integral subunit II from Cyt-D to the Cu(A) redox center most likely involves the iron ligand histidine 20 as an electron exit point in Cyt-D.

Evidence for the presence of two conformations of the heme a3-Cu(B) pocket of cytochrome caa3 from Thermus thermophilus.

Resonance Raman (RR) and "light" minus "dark" Fourier transform infrared (FTIR) difference spectra are reported for the CO-bound caa(3) oxidase from Thermus thermophilus. Two Fe-CO stretching modes at 518 and 507 cm(-1), the Fe-C-O bending mode at 570 cm(-1), and three C-O modes of heme a(3) at 1958, 1967, and 1973 cm(-1) have been identified in the RR and FTIR spectra, respectively. The FTIR "light" minus "dark" spectrum indicates the formation of Cu(B)CO as revealed by its ν(CO) at 2060/2065 cm(-1). We assign the bands at 518 (ν(Fe-CO)) and 1967/1973 cm(-1) (ν(C-O)) as the α-conformation. We also assign the bands at 507 and 1958 cm(-1) (ν(C-O)) as originating from the ß-conformation of the enzyme. A frequency upshift of the heme a(3) Fe-His mode is observed subsequent to CO photolysis from 209 cm(-1) in the equilibrium deoxy enzyme to 214 cm(-1) in the photoproduct. The caa(3) data, distinctly different from those of ba(3) oxidase, are discussed in terms of the coupling of the α- and ß-conformations that occur in heme-copper oxidases with catalytic function. The dynamics between the heme a(3) and heme a propionates as revealed by the perturbation of the bending vibrations δ(prop) of hemes a and a(3) at 385 and 392 cm(-1), respectively, induced upon CO binding to heme a(3) is discussed in terms of the protonic connectivity between the heme a ring-D propionate/Arg site with that of the heme a(3) ring-D propionate-H(2)O site that leads to the highly conserved in the heme-copper oxidases water pool.

The alternative complex III of Rhodothermus marinus and its structural and functional association with caa3 oxygen reductase.

An alternative complex III (ACIII) is a respiratory complex with quinol:electron acceptor oxidoreductase activity. It is the only example of an enzyme performing complex III function that does not belong to bc1 complex family. ACIII from Rhodothermus (R.) marinus was the first enzyme of this type to be isolated and characterized, and in this work we deepen its characterization. We addressed its interaction with quinol substrate and with the caa3 oxygen reductase, whose coding gene cluster follows that of the ACIII. There is at least, one quinone binding site present in R. marinus ACIII as observed by fluorescence quenching titration of HQNO, a quinone analogue inhibitor. Furthermore, electrophoretic and spectroscopic evidences, taken together with mass spectrometry revealed a structural association between ACIII and caa3 oxygen reductase. The association was also shown to be functional, since quinol:oxygen oxidoreductase activity was observed when the two isolated complexes were put together. This work is thus a step forward in the recognition of the structural and functional diversities of prokaryotic respiratory chains.

Thermodynamic redox behavior of the heme centers in A-type heme-copper oxygen reductases: comparison between the two subfamilies.

The study of the thermodynamic redox behavior of the hemes from two members of the A family of heme-copper oxygen reductases, Paracoccus denitrificans aa3 (A1 subfamily) and Rhodothermus marinus caa3 (A2 subfamily) enzymes, is presented. At different pH values, midpoint reduction potentials and interaction potentials were obtained in the framework of a pairwise model for two interacting redox centers. In both enzymes, the hemes have different reduction potentials. For the A1-type enzyme, it was shown that heme a has a pH-dependent midpoint reduction potential, whereas that of heme a3 is pH independent. For the A2-type enzyme the opposite was observed. The midpoint reduction potential of heme c from subunit II of the caa3 enzyme was determined by fitting the data with a single-electron Nernst curve, and it was shown to be pH dependent. The results presented here for these A-type enzymes are compared with those previously obtained for representative members of the B and C families.

Study of redox potential in cytochrome c covalently bound to terminal oxidase of alkaliphilic Bacillus pseudofirmus FTU.

Spectroelectrochemistry was used to determine the midpoint redox potentials of heme cofactors of the caa3-type cytochrome oxidase from the alkaliphilic bacterium Bacillus pseudofirmus FTU. The apparent midpoint potentials (E(m)(app)) for the most prominent transitions of hemes a and a3 (+193 and +334 mV, respectively) were found to be similar to the values reported for other enzymes with high homology to the caa3-type oxidase. In contrast, the midpoint potential of the covalently bound cytochrome c (+89 mV) was 150-170 mV lower than in cytochromes c, either low molecular weight or covalently bound to the caa3 complex in all known aerobic neutralophilic and thermo-neutralophilic bacteria. Such an unusually low redox potential of the covalently bound cytochrome c of the caa3-type oxidase of alkaliphilic bacteria, together with high redox potentials of hemes a and a3, ensures more than twice higher difference in redox potentials inside the respiratory complex compared to the homologous mitochondrial enzyme. The energy released during this redox transition might be stored in the transmembrane H+ gradient even under low Deltap in the alkaline environment of the bacteria at the expense of a significant increase in DeltaG of the coupled redox reaction.

Electron transfer kinetics of soluble fragments indicate a direct interaction between complex III and the caa3 oxidase in Thermus thermophilus.

The extremely thermophilic bacterium Thermus thermophilus expresses an aerobic respiratory chain resembling that of mitochondria and many mesophilic prokaryotes. Yet, interaction modes between redox partners differ between the thermophilic and mesophilic electron transport chains. While electron transfer in mesophilic organisms such as Paracoccus denitrificans follows a two-step mechanism mostly governed by long-range electrostatic interactions, the electron transfer in thermophiles is mediated mainly by apolar interactions. The terminal branch of the electron path from the bc-complex via the soluble cytochrome c(552) to the ba(3) oxidase has extensively been characterized, whereas contradicting evidence has been put forward on the nature of the physiological substrate(s) of the caa(3) oxidase. We have cloned and expressed a soluble fragment of the hydrophilic cytochrome c domain derived from subunit IIc of the caa(3) oxidase (c(caa)(3)) and characterized its kinetic behaviour in terms of substrate specificity and ionic strength dependency using pre-steady state stopped-flow techniques. The kinetics revealed fast electron transfer between the caa(3) fragment and both, the cytochrome c(552) and the soluble cytochrome c(bc) fragment of the bc-complex, showing only a weak ionic strength dependence. These data suggest a direct intercomplex electron transfer between the bc-complex and the caa(3) oxidase without requirement for a soluble electron shuttle.

Rat carotid body chemosensory discharge and glomus cell HIF-1 alpha expression in vitro: regulation by a common oxygen sensor.

Addition of Pco ( approximately 350 Torr) to a normoxic medium (Po(2) of approximately 130 Torr) was used to investigate the relationship between carotid body (CB) sensory discharge and expression of hypoxia-inducible factor 1 alpha (HIF-1 alpha) in glomus cells. Afferent electrical activity measured for in vitro-perfused rat CB increased rapidly (1-2 s) with addition of high CO (Pco of approximately 350 Torr; Po(2) of approximately 130 Torr), and this increase was fully reversed by white light. At submaximal light intensities, the extent of reversal was much greater for monochromatic light at 430 and 590 nm than for light at 450, 550, and 610 nm. This wavelength dependence is consistent with the action spectrum of the CO compound of mitochondrial cytochrome a(3). Interestingly, when isolated glomus cells cultured for 45 min in the presence of high CO (Pco of approximately 350 Torr; Po(2) of approximately 130 Torr) in the dark, the levels of HIF-1 alpha, which turn over slowly (many minutes), increased. This increase was not observed if the cells were illuminated with white light during the incubation. Monochromatic light at 430- and 590-nm light was much more effective than that at 450, 550, and 610 nm in blocking the CO-induced increase in HIF-1 alpha, as was the case for chemoreceptor discharge. Although the changes in HIF-1 alpha take minutes and those for CB neural activity occur in 1-2 s, the similar responses to CO and light suggest that the oxygen sensor is the same (mitochondrial cytochrome a(3)).

Interaction between cytochrome caa3 and F1F0-ATP synthase of alkaliphilic Bacillus pseudofirmus OF4 is demonstrated by saturation transfer electron paramagnetic resonance and differential scanning calorimetry assays.

Interaction between the cytochrome caa3 respiratory chain complex and F1F0-ATP synthase from extremely alkaliphilic Bacillus pseudofirmus OF4 has been hypothesized to be required for robust ATP synthesis by this alkaliphile under conditions of very low protonmotive force. Here, such an interaction was probed by differential scanning calorimetry (DSC) and by saturation transfer electron paramagnetic resonance (STEPR). When the two purified complexes were embedded in phospholipid vesicles individually [(caa3)PL, (F1F0)PL)] or in combination [(caa3 + F1F0)PL] and subjected to DSC analysis, they underwent exothermic thermodenaturation with transition temperatures at 69, 57, and 46/75 degrees C, respectively. The enthalpy change, deltaH (-8.8 kcal/mmol), of protein-phospholipid vesicles containing both cytochrome caa3 and F1F0 was smaller than that (-12.4 kcal/mmol) of a mixture of protein-phospholipid vesicles formed from each individual electron transfer complex [(caa3)PL + (F1F0)PL]. The rotational correlation time of spin-labeled caa3 (65 micros) in STEPR studies increased significantly when the complex was mixed with F1F0 prior to being embedded in phospholipid vesicles (270 micros). When the complexes were reconstituted separately and then mixed together, or either mitochondrial cytochrome bc1 or F1F0 was substituted for the alkaliphile F1F0, the correlation time was unchanged (65-70 micros). Varying the ratio of the two alkaliphile complexes in both the DSC and STEPR experiments indicated that the optimal stoichiometry is 1:1. These results demonstrate a physical interaction between the cytochrome caa3 and F1F0-ATP synthase from B. pseudofirmus OF4 in a reconstituted system. They support the suggestion that such an interaction between these complexes may contribute to sequestered proton transfers during alkaliphile oxidative phosphorylation at high pH.

Cytochrome c oxidase maintains mitochondrial respiration during partial inhibition by nitric oxide.

Nitric oxide (NO), generated endogenously in NO-synthase-transfected cells, increases the reduction of mitochondrial cytochrome c oxidase (CcO) at O2 concentrations ([O2]) above those at which it inhibits cell respiration. Thus, in cells respiring to anoxia, the addition of 2.5 microM L-arginine at 70 microM O2 resulted in reduction of CcO and inhibition of respiration at [O2] of 64.0+/-0.8 and 24.8+/-0.8 microM, respectively. This separation of the two effects of NO is related to electron turnover of the enzyme, because the addition of electron donors resulted in inhibition of respiration at progressively higher [O2], and to their eventual convergence. Our results indicate that partial inhibition of CcO by NO leads to an accumulation of reduced cytochrome c and, consequently, to an increase in electron flux through the enzyme population not inhibited by NO. Thus, respiration is maintained without compromising the bioenergetic status of the cell. We suggest that this is a physiological mechanism regulated by the flux of electrons in the mitochondria and by the changing ratio of O2:NO, either during hypoxia or, as a consequence of increases in NO, as a result of cell stress.

A tyrosine residue deprotonates during oxygen reduction by the caa3 reductase from Rhodothermus marinus.

Heme-copper oxygen reductases catalyze proton translocation across the cellular membrane; this takes place during the reaction of oxygen to water. We demonstrate with attenuated total reflection-Fourier transform infrared (ATR-FTIR) difference spectroscopy that a tyrosine residue of the oxygen reductase from the thermohalophilic Rhodothermus marinus becomes deprotonated in the transition from the oxidized state to the catalytic intermediate ferryl state P(M). This tyrosine residue is most probably Y256, the helix VI tyrosine residue proposed to substitute for the D-channel glutamic acid that is absent in this enzyme. Comparison with the mitochondrial like oxygen reductase from Rhodobacter sphaeroides suggests that proton transfer from a strategically situated donor to the active site is a crucial step in the reaction mechanism of oxygen reductases.

Expression, purification, and characterization of the CuA-cytochrome c domain from subunit II of the Bacillus subtilis cytochrome caa3 complex in Escherichia coli.

Cytochrome caa3 from Bacillus subtilis is a member of the heme-copper oxidase family of integral membrane enzymes that includes mitochondrial cytochrome c oxidase. Subunit II of cytochrome caa3 has an extra 100 amino acids at its C-terminus, relative to its mitochondrial counterpart, and this extension encodes a heme C binding domain. Cytochrome caa3 has many of the properties of the complex formed between mitochondrial cytochrome c and mitochondrial cytochrome c oxidase. To examine more closely the interaction between cytochrome c and the oxidase we have cloned and expressed the Cu(A)-cytochrome c portion of subunit II from the cytochrome caa3 complex of B. subtilis. We are able to express about 2000 nmol, equivalent to 65 mg, of the Cu(A)-cytochrome c protein per litre of Escherichia coli culture. About 500 nmol is correctly targeted to the periplasmic space and we purify 50% of that by a combination of affinity chromatography and ammonium sulfate fractionation. The cytochrome c containing sub-domain is well-folded with a stable environment around the heme C center, as its mid-point potential and rates of reduction are indistinguishable from values for the cytochrome c domain of the holo-enzyme. However, the Cu(A) site lacks copper leading to an inherent instability in this sub-domain. Expression of B. subtilis cytochrome c, as exemplified by the Cu(A)-cytochrome c protein, can be achieved in E. coli, and we conclude that the cytochrome c and Cu(A) sub-domains behave independently despite their close physical and functional association.

Structure at 1.3 A resolution of Rhodothermus marinus caa(3) cytochrome c domain.

The cytochrome c domain of subunit II from the Rhodothermus marinus caa(3) HiPIP:oxygen oxidoreductase, a member of the superfamily of heme-copper-containing terminal oxidases, was produced in Escherichia coli and characterised. The recombinant protein, which shows the same optical absorption and redox properties as the corresponding domain in the holo enzyme, was crystallized and its structure was determined to a resolution of 1.3 A by the multiwavelength anomalous dispersion (MAD) technique using the anomalous dispersion of the heme iron atom. The model was refined to final R(cryst) and R(free) values of 13.9% and 16.7%, respectively. The structure reveals the insertion of two short antiparallel beta-strands forming a small beta-sheet, an interesting variation of the classical all alpha-helical cytochrome c fold. This modification appears to be common to all known caa(3)-type terminal oxidases, as judged by comparative modelling and by analyses of the available amino acid sequences for these enzymes. This is the first high-resolution crystal structure reported for a cytochrome c domain of a caa(3)-type terminal oxidase. The R.marinus caa(3) uses HiPIP as the redox partner. The calculation of the electrostatic potential at the molecular surface of this extra C-terminal domain provides insights into the binding to its redox partner on one side and its interaction with the remaining subunit II on the other side.