NO binding and dynamics in reduced heme-copper oxidases aa3 from Paracoccus denitrificans and ba3 from Thermus thermophilus

Cytochrome c oxidase (CcO) has a high affinity for nitric oxide (NO), a property involved in the regulation of respiration. It has been shown that the recombination kinetics of photolyzed NO with reduced CcO from Paracoccus denitrificans on the picosecond time scale depend strongly on the NO/enzyme stoichiometry and inferred that more than one NO can be accommodated by the active site, already at mildly suprastoichiometric NO concentrations. We have largely extended these studies by monitoring rebinding dynamics from the picosecond to the microsecond time scale, by performing parallel steady-state low-temperature electron paramagnetic resonance (EPR) characterizations on samples prepared similarly as for the optical experiments and comparing them with molecular-modeling results. A comparative study was performed on CcO ba(3) from Thermus thermophilus, where two NO molecules cannot be copresent in the active site in the steady state because of its NO reductase activity. The kinetic results allow discrimination between different models of NO-dependent recombination and show that the overall NO escape probability out of the protein is high when only one NO is bound to CcO aa(3), whereas strong rebinding on the 15-ns time scale was observed for CcO ba(3). The EPR characterizations show similar results for aa(3) at substoichiometric NO/enzyme ratios and for ba(3), indicating formation of a 6-coordinate heme-NO complex. The presence of a second NO molecule in the aa(3) active site strongly modifies the heme-NO EPR spectrum and can be rationalized by a rotation of the Fe-N-O plane with respect to the histidine that coordinates the heme iron. This proposal is supported by molecular-modeling studies that indicate a approximately 63 degrees rotation of heme-bound NO upon binding of a second NO to the close-lying copper center CuB. It is argued that the second NO binds to CuB.     

The mixed valence state of the oxidase binuclear centre: how Thermus thermophilus cytochrome ba3 differs from classical aa3 in the aerobic steady state and when inhibited by cyanide

In the aerobic steady state of the classical eukaryotic cytochrome c oxidase, three aa(3) redox metal centres (cytochrome a, CuA and CuB) are partially reduced while the fourth, cytochrome a(3), remains almost fully oxidized. Turnover depends primarily upon the rate of cytochrome a(3) reduction. When prokaryotic cytochrome c-552 oxidase (ba(3)) of Thermus thermophilus turns over, three different metal centres (cytochromes b, a(3) and CuA) share the steady state electrons; it is the fourth, CuB, that apparently remains almost fully oxidized until anaerobiosis. Cytochrome a(3) stays partially reduced during turnover and a possible P/F state may also be populated. Cyanide traps the aerobic ba(3) CuB centre in the a(3)(2+)CNCuB(2+) state; the corresponding eukaryotic cyanide trapped state is a(3)(3+)CNCuB(+). Both states become the fully reduced a(3)(2+)CNCuB(+) upon anaerobiosis. The different reactivities of the aa(3) and ba(3) binuclear centres may be correlated with the very different proximal histidine N-Fe distances in the two enzymes (3.3 A for ba(3) compared to 1.9 A for aa(3)) which may in turn relate to the functioning of thermophilic Thermus cytochrome ba(3) in vivo at a very elevated temperature. But the differences may also just exemplify how evolution can find surprisingly different solutions to the common problem of electron transfer to oxygen. Some of these alternatives were potentially enshrined in a model of the oxidase reaction already adopted by Gerry Babcock in the early 1990s.     

Exploring the cytochrome c folding mechanism: cytochrome c552 from thermus thermophilus folds through an on-pathway intermediate

Understanding the role of partially folded intermediate states in the folding mechanism of a protein is a crucial yet very difficult problem. We exploited a kinetic approach to demonstrate that a transient intermediate of a thermostable member of the widely studied cytochrome c family (cytochrome c552 from Thermus thermophilus) is indeed on-pathway. This is the first clear indication of an obligatory intermediate in the folding mechanism of a cytochrome c. The fluorescence properties of this intermediate demonstrate that the relative position of the heme and of the only tryptophan residue cannot correspond to their native orientation. Based on an analysis of the three-dimensional structure of cytochrome c552, we propose an interpretation of the data which explains the residual fluorescence of the intermediate and is consistent with the established role played by some conserved interhelical interactions in the folding of other members of this family. A limited set of topologically conserved contacts may guide the folding of evolutionary distant cytochromes c through the same partially structured state, which, however, can play different kinetic roles, acting either as an intermediate or a transition state.     

Observation of the equilibrium CuB-CO complex and functional implications of the transient heme a3 propionates in cytochrome ba3-CO from Thermus thermophilus. Fourier transform infrared (FTIR) and time-resolved step-scan FTIR studies

We report the first evidence for the existence of the equilibrium Cu(B)1+-CO species of CO-bound reduced cytochrome ba(3) from Thermus thermophilus at room temperature. The frequency of the C-O stretching mode of Cu(B)1+-CO is located at 2053 cm(-1) and remains unchanged in H(2)O/D(2)O exchanges and, between pD 5.5 and 9.7, indicating that the chemical environment does not alter the protonation state of the Cu(B) histidine ligands. The data and conclusions reported here are in contrast to the changes in protonation state of Cu(B)-His-290, reported recently (Das, T. K., Tomson, F. K., Gennis, R. B., Gordon, M., and Rousseau, D. L. (2001) Biophys. J. 80, 2039-2045 and Das, T. P., Gomes, C. M., Teixeira, M., and Rousseau, D. L. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 9591-9596). The time-resolved step-scan FTIR difference spectra indicate that the rate of decay of the transient Cu(B)1+-CO complex is 34.5 s(-1) and rebinding to heme a(3) occurs with k(2) = 28.6 s(-1). The rate of decay of the transient Cu(B)1+-CO complex displays a similar time constant as the absorption changes at 1694(+)/1706(-), attributed to perturbation of the heme a(3) propionates (COOH). The nu(C-O) of the transient Cu(B)1+-CO species is the same as that of the equilibrium Cu(B)1+-CO species and remains unchanged in the pD range 5.5-9.7 indicating that no structural change takes place at Cu(B) between these states. The implications of these results with respect to proton pathways in heme-copper oxidases are discussed.     

Microextraction of bacterial lipid A: easy and rapid method for mass spectrometric characterization

Endotoxins (lipopolysaccharides) are the main components of Gram-negative bacterial outer membranes. A quick and simple way to isolate their lipid region (lipid A) directly from whole bacterial cells was devised. This method using hot ammonium-isobutyrate solvent was applied to small quantities of cells and proved to be indispensable when a rapid characterization of lipid A structure by mass spectrometry was required. Biological activities of endotoxins are directly related to the lipid A structures, which vary greatly with cell growth conditions. This method is suitable for rough- and smooth-type bacteria and very efficient for screening variations in lipid A structures. Data are acquired in a few hours and avoid the use of phenol in extraction.     

A novel apolipoprotein A-1 variant, Arg173Pro, associated with cardiac and cutaneous amyloidosis

An American kindred was found to have hereditary amyloidosis with cutaneous and cardiac involvement. Characterization of fibrils isolated from skin identified the amyloid protein as the N-terminal 90 to 100 residues of apolipoprotein A-1. Sequence of the apolipoprotein A-1 gene was normal except for a G/C transversion at position 1638 which predicts an Arg to Pro substitution at residue 173. This mutation, unlike previously described amyloidogenic mutations is not in the N-terminal fragment which is incorporated into the fibril. The mutation is at the same residue as in apolipoprotein A-1 Milano (Arg173Cys) which does not result in amyloid formation. Decreased plasma HDL cholesterol levels in carriers of the Arg173Pro mutation suggest an increased rate of catabolism as has been shown for the amyloidogenic Gly26Arg mutation. This suggests that altered metabolism caused by the mutation may be a significant factor in apolipoprotein A-1 fibrillogenesis.     

High thermal and chemical stability of Thermus thermophilus seven-iron ferredoxin. Linear clusters form at high pH on polypeptide unfolding.

To probe the stability of the seven-iron ferredoxin from Thermus thermophilus (FdTt), we investigated its chemical and thermal denaturation processes in solution. As predicted from the crystal structure, FdTt is extremely resistant to perturbation. The guanidine hydrochloride-induced unfolding transition shows a midpoint at 6.5 m (pH 7, 20 degrees C), and the thermal midpoint is above boiling, at 114 degrees C. The stability of FdTt is much lower at acidic pH, suggesting that electrostatic interactions are important for the high stability at higher pH. On FdTt unfolding at alkaline pH, new absorption bands at 520 nm and 610 nm appear transiently, resulting from rearrangement of the cubic clusters into linear three-iron species. A range of iron-sulfur proteins has been found to accommodate these novel clusters in vitro, although no biological function has yet been assigned.     

Regulation of S-adenosyl methionine synthesis in the mouse embryo

In early embryos, methylation is involved in "gamete imprinting" and inactivation of artificially introduced foreign genes. We studied the biosynthesis of the universal methylation cofactor: S-Adenosyl methionine (SAM). In the mouse, SAM conversion from methionine is limited by saturation of the methionine endogenous pool. SAM is present at a practically unchanged level from the unfertilized oocyte to early morula. SAM synthesis is increased at the time of compaction. In blastocysts, although methionine uptake is increased, the conversion rate from methionine is lowered. We observed no differences between C57 Black and Swiss albino random bred strains. In few experiments with human unfertilized oocytes and spared embryos, we observed higher methionine incorporation, and higher conversion to SAM. Next, the effect of two methylation inhibitors was tested, on early mouse embryonic development, at the one-cell and the two-cell stage. We found that ethionine is very toxic, even at the lowest tested concentration of 25 microM. Homocysteine is more potent at the one-cell stage than at the 2-cell stage, and it only partially blocks blastocyst formation from the 2-cell stage even at a concentration of 500 microM. It clearly acts as a methylation inhibitor; it lowers the SAM pool and the methylation index, SAH/SAM ratio (SAH: S-Adenosyl Homocysteine). We also found that homocysteine is an unexpected competitor for methionine influx and efflux.     

The Murine Prepuberal Oviduct Supports Early Embryo Development In Vitro

Swiss albino mice were used to evaluate the ability of explanted murine oviducts to support development through the block stage. One cell eggs develop as well when cultured 50 hours in oviducts explanted from pregnant mice or in oviducts from immature mice: The blastocyst formation occurs at a similar rate in both cases. The viability of the blastocysts was high (8/9) when transferred to pseudopregnant (C 57 Black) recipient mice. Only a few difference was observed in the polypeptide pattern of immature and pregnant explanted oviduct. In immature oviduct, the polypeptide secretory profile is not modified by the presence of fertilized embryos transferred into it, and so nor is it directly egg dependent. It is concluded that oviduct's ability to sustain normal early embryonic developments is not dependent upon sexual maturity.     

The distribution pattern of chlorogenic acid and caffeine inCoffea arabica

Zusammenfassung Im Hinblick auf die vielfach erwähnte Komplexbildung (Salzbildung) zwischen Kaffein und Chlorogensäure wurden alle vegetativen Teile und Früchte von jungen Kaffeebäumen auf das Mengenverhältnis der beiden Substanzen untersucht. Der Gehalt an Chlorogensäure ist in allen Organen höher als der Kaffeingehalt. Die Variation des Gehaltes mit dem Blattalter ist nicht gleich für die zwei Substanzen, das Maximum des Chlorogensäuregehaltes wird in einem früheren Entwicklungsstadium erreicht als dasjenige des Kaffeingehaltes. Es scheint unwahrscheinlich, daß die in vitro feststellbare Bindung zwischen Kaffein und Chlorogensäure stoffwechselphysiologisch eine Rolle spielt.

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