Atopy, asthma, and lung function in relation to folate and vitamin B(12) in adults.

BACKGROUND: Recent studies suggested low serum folate and impaired folate metabolism as potential risk factors for development of asthma and atopic disease, but the results are inconsistent. The aim of this study was to investigate the relations of markers of folate and vitamin B12 (B12) deficiency with different phenotypes of asthma and atopy. METHODS: A random sample of 6784 persons from a general population aged 30-60 years participated in a health examination in 1999-2001, and 4516 (66.6%) of those also participated in a follow-up examination 5 years later. The examinations included spirometry, measurements of serum folate and B12, specific IgE to inhalant allergens, total IgE, and genotyping of the MTHFR-C677T polymorphism - a genetic marker of impaired folate metabolism. Information about dietary intake of folate and B12, asthma diagnosis, and airway symptoms was obtained by questionnaires. RESULTS: Low serum folate levels and the TT genotype of the MTHFR-C677T polymorphism were associated with increased prevalence of self-reported doctor-diagnosed asthma [odds ratio (OR) 1.37, 95% confidence interval (CI) 1.05-1.79 and OR 1.52; 95% CI 1.12-2.06, respectively] and attacks of shortness of breath (OR 1.43, 95% CI 1.14-1.79 and OR 1.47; 95% CI: 1.14-1.91, respectively). We found no significant associations with lung function or atopic outcomes. Serum levels of B12 and dietary intake of folate and B12 were not associated with asthma or atopy. CONCLUSIONS: We found that two objective markers of folate deficiency were associated with self-reported doctor-diagnosed asthma and attacks of shortness of breath, but not with lung function or atopy.

Kinetics of electron and proton transfer during O(2) reduction in cytochrome aa(3) from A. ambivalens: an enzyme lacking Glu(I-286).

Acidianus ambivalens is a hyperthermoacidophilic archaeon which grows optimally at approximately 80 degrees C and pH 2.5. The terminal oxidase of its respiratory system is a membrane-bound quinol oxidase (cytochrome aa(3)) which belongs to the heme-copper oxidase superfamily. One difference between this quinol oxidase and a majority of the other members of this family is that it lacks the highly-conserved glutamate (Glu(I-286), E. coli ubiquinol oxidase numbering) which has been shown to play a central role in controlling the proton transfer during reaction of reduced oxidases with oxygen. In this study we have investigated the dynamics of the reaction of the reduced A. ambivalens quinol oxidase with O(2). With the purified enzyme, two kinetic phases were observed with rate constants of 1.8&z.ccirf;10(4) s(-1) (at 1 mM O(2), pH 7.8) and 3. 7x10(3) s(-1), respectively. The first phase is attributed to binding of O(2) to heme a(3) and oxidation of both hemes forming the 'peroxy' intermediate. The second phase was associated with proton uptake from solution and it is attributed to formation of the 'oxo-ferryl' state, the final state in the absence of quinol. In the presence of bound caldariella quinol (QH(2)), heme a was re-reduced by QH(2) with a rate of 670 s(-1), followed by transfer of the fourth electron to the binuclear center with a rate of 50 s(-1). Thus, the results indicate that the quinol donates electrons to heme a, followed by intramolecular transfer to the binuclear center. Moreover, the overall electron and proton-transfer kinetics in the A. ambivalens quinol oxidase are the same as those in the E. coli ubiquinol oxidase, which indicates that in the A. ambivalens enzyme a different pathway is used for proton transfer to the binuclear center and/or other protonatable groups in an equivalent pathway are involved. Potential candidates in that pathway are two glutamates at positions (I-80) and (I-83) in the A. ambivalens enzyme (corresponding to Met(I-116) and Val(I-119), respectively, in E. coli cytochrome bo(3)).

Redesign of the proton-pumping machinery of cytochrome c oxidase: proton pumping does not require Glu(I-286).

One of the putative proton-transfer pathways leading from solution toward the binuclear center in many cytochrome c oxidases is the D-pathway, so-called because it starts with a highly conserved aspartate [D(I-132)] residue. Another highly conserved amino acid residue in this pathway, glutamate(I-286), has been indicated to play a central role in the proton-pumping machinery of mitochondrial-type enzymes, a role that requires a movement of the side chain between two distinct positions. In the present work we have relocated the glutamate to the opposite side of the proton-transfer pathway by constructing the double mutant EA(I-286)/IE(I-112). This places the side chain in about the same position in space as in the original enzyme, but does not allow for the same type of movement. The results show that the introduction of the second-site mutation, IE(I-112), in the EA(I-286) mutant enzyme results in an increase of the enzyme activity by a factor of >10. In addition, the double mutant enzyme pumps approximately 0.4 proton per electron. This observation restricts the number of possible mechanisms for the operation of the redox-driven proton pump. The proton-pumping machinery evidently does require the presence of a protonatable/polar residue at a specific location in space, presumably to stabilize an intact water chain. However, this residue does not necessarily have to be at a strictly conserved location in the amino acid sequence. In addition, the results indicate that E(I-286) is not the "proton gate" of cytochrome c oxidase controlling the flow of pumped protons from one to the other side of the membrane.

Proton transfer from glutamate 286 determines the transition rates between oxygen intermediates in cytochrome c oxidase.

We have investigated the electron-proton coupling during the peroxy (P(R)) to oxo-ferryl (F) and F to oxidised (O) transitions in cytochrome c oxidase from Rhodobacter sphaeroides. The kinetics of these reactions were investigated in two different mutant enzymes: (1) ED(I-286), in which one of the key residues in the D-pathway, E(I-286), was replaced by an aspartate which has a shorter side chain than that of the glutamate and, (2) ML(II-263), in which the redox potential of Cu(A) is increased by approximately 100 mV, which slows electron transfer to the binuclear centre during the F-->O transition by a factor of approximately 200. In ED(I-286) proton uptake during P(R)-->F was slowed by a factor of approximately 5, which indicates that E(I-286) is the proton donor to P(R). In addition, in the mutant enzyme the F-->O transition rate displayed a deuterium isotope effect of approximately 2.5 as compared with approximately 7 in the wild-type enzyme. Since the entire deuterium isotope effect was shown to be associated with a single proton-transfer reaction in which the proton donor and acceptor must approach each other (M. Karpefors, P. Adelroth, P. Brzezinski, Biochemistry 39 (2000) 6850), the smaller deuterium isotope effect in ED(I-286) indicates that proton transfer from E(I-286) determines the rate also of the F-->O transition. In ML(II-263) the electron-transfer to the binuclear centre is slower than the intrinsic proton-transfer rate through the D-pathway. Nevertheless, both electron and proton transfer to the binuclear centre displayed a deuterium isotope effect of approximately 8, i.e., about the same as in the wild-type enzyme, which shows that these reactions are intimately coupled.

Dynamics of the binuclear center of the quinol oxidase from Acidianus ambivalens.

We have investigated the kinetic and thermodynamic properties of carbon monoxide binding to the fully reduced quinol oxidase (cytochrome aa(3)) from the hyperthermophilic archaeon Acidianus ambivalens. After flash photolysis of CO from heme a(3), the complex recombines with an apparent rate constant of approximately 3 s(-1), which is much slower than with the bovine cytochrome c oxidase (approximately 80 s(-1)). Investigation of the CO-recombination rate as a function of the CO concentration shows that the rate saturates at high CO concentrations, which indicates that CO must bind transiently to Cu(B) before binding to heme a(3). With the A. ambivalens enzyme the rate reached 50% of its maximum level (which reflects the dissociation constant of the Cu(B)(CO) complex) at approximately 13 microM CO, which is a concentration approximately 10(3) times smaller than for the bovine enzyme (approximately 11 mM). After CO dissociation we observed a rapid absorbance relaxation with a rate constant of approximately 1.4 x 10(4) s(-1), tentatively ascribed to a heme-pocket relaxation associated with release of CO after transient binding to Cu(B). The equilibrium constant for CO transfer from Cu(B) to heme a(3) was approximately 10(4) times smaller for the A. ambivalens than for the bovine enzyme. The approximately 10(3) times smaller Cu(B)(CO) dissociation constant, in combination with the approximately 10(4) times smaller equilibrium constant for the internal CO transfer, results in an apparent dissociation constant of the heme a(3)(CO) complex which is "only" about 10 times larger for the A. ambivalens ( approximately 4 x 10(-3) mM) than for the bovine (0.3 x 10(-3) mM) enzyme. In summary, the results show that while the basic mechanism of CO binding to the binuclear center is similar in the A. ambivalens and bovine (and R. sphaeroides) enzymes, the heme-pocket dynamics of the two enzymes are dramatically different, which is discussed in terms of the different structural details of the A. ambivalens quinol oxidase and adaptation to different living conditions.