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Substrate Control of Internal Electron Transfer in Bacterial Nitric-oxide Reductase
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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2010 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, no 33, 25531-25537 p.Article in journal (Refereed) Published
Abstract [en]

Nitric-oxide reductase (NOR) from Paracoccus denitrificans catalyzes the reduction of nitric oxide (NO) to nitrous oxide (N2O) (2NO + 2H(+) + 2e(-) -> N2O + H2O) by a poorly understood mechanism. NOR contains two low spin hemes c and b, one high spin heme b(3), and a non-heme iron Fe-B. Here, we have studied the reaction between fully reduced NOR and NO using the ""flow-flash"" technique. Fully (four-electron) reduced NOR is capable of two turnovers with NO. Initial binding of NO to reduced heme b(3) occurs with a time constant of similar to 1 mu s at 1.5 mM NO, in agreement with earlier studies. This reaction is [NO]-dependent, ruling out an obligatory binding of NO to FeB before ligation to heme b(3). Oxidation of hemes b and c occurs in a biphasic reaction with rate constants of 50 s(-1) and 3 s(-1) at 1.5 mM NO and pH 7.5. Interestingly, this oxidation is accelerated as [NO] is lowered; the rate constants are 120 s(-1) and 12 s(-1) at 75 mu M NO. Protons are taken up from solution concomitantly with oxidation of the low spin hemes, leading to an acceleration at low pH. This effect is, however, counteracted by a larger degree of substrate inhibition at low pH. Our data thus show that substrate inhibition in NOR, previously observed during multiple turnovers, already occurs during a single oxidative cycle. Thus, NO must bind to its inhibitory site before electrons redistribute to the active site. The further implications of our data for the mechanism of NO reduction by NOR are discussed.

Place, publisher, year, edition, pages
2010. Vol. 285, no 33, 25531-25537 p.
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-50090DOI: 10.1074/jbc.M110.123984ISI: 000280682400047OAI: oai:DiVA.org:su-50090DiVA: diva2:382297
Note

authorCount :5

Available from: 2010-12-30 Created: 2010-12-21 Last updated: 2017-12-11Bibliographically approved
In thesis
1. Proton transfer in nitric oxide reducing heme-copper oxidases
Open this publication in new window or tab >>Proton transfer in nitric oxide reducing heme-copper oxidases
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Heme-copper oxidases (HCuOs) are best known as terminal oxidases in the aerobic respiratory chain, in which they catalyze the reduction of oxygen to water. By receiving protons and electrons from opposite sides of the membrane as well as pumping protons, HCuOs contribute to the electrochemical proton gradient over the membrane that can be used for ATP synthesis. Divergent members of the HCuO superfamily are nitric oxide reductases (NORs) that catalyze the reduction of nitric oxide (NO) to nitrous oxide (N2O) as part of the denitrification process, an alternative respiratory pathway.

The first part of the thesis focuses on electron and proton transfer reactions that are associated with the reductive conversion of NO to N2O and O2 to H2O by the NOR from Paracoccus denitrificans. Our data show that proton uptake in NOR is not electrogenic (protons and electrons are taken up from the same side of the membrane) and that no protons are pumped. Also, structural variants have been investigated and the results suggest a role for these residues in proton transfer. Further, we show that lowering the pH leads to a higher NO reduction rate, while this effect is partially counteracted by a larger degree of substrate inhibition at low pH.

The second part deals with proton transfer and electrical potential generation in the reaction between the cbb3 oxidase from Rhodobacter sphaeroides and O2 or NO. Our data show that NO reduction by cbb3 oxidase is not coupled to proton translocation and that the direction of proton uptake is dependent on substrate. Our findings suggest that the proton pumping mechanism in HCuOs is incompatible with NO reduction intermediates.

Finally, experiments on structural variants of the ba3 oxidase from Thermus thermophilus indicate a functional role for the inspected residues in proton transfer and support the suggestion that a single proton-transfer pathway is used in the ba3 oxidase.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2011. 76 p.
Keyword
heme-copper oxidases, nitric oxide, proton transfer, electron transfer, proton-transfer pathway
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-62893 (URN)978-91-7447-377-3 (ISBN)
Public defence
2011-11-11, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 14:00 (English)
Opponent
Supervisors
Available from: 2011-10-20 Created: 2011-10-03 Last updated: 2011-10-17Bibliographically approved
2. Kinetics of proton and electron transfer in heme-copper oxidases
Open this publication in new window or tab >>Kinetics of proton and electron transfer in heme-copper oxidases
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Heme-copper oxidases are transmembrane proteins that are found in aerobic and anaerobic respiratory chains. During aerobic respiration, these enzymes reduce dioxygen to water. The energy released in the reaction is used to transport protons across a biological membrane. Stored as proton electrochemical gradient, the energy can be used to regenerate ATP. It is known that aa3 oxidases, which are the most common oxidases, transport pumped protons and protons used for the catalytic reaction using two proton pathways. However, the molecular mechanism of pumping is still being debated.

When oxygen is available in very small quantities, oxygen reductases with high affinity for oxygen are expressed by organisms like Thermus thermophilus. The proton pumping mechanism in the ba3 oxidase is slightly different from that of aa3 oxidases as this enzyme only uses a single proton uptake pathway. Here we analyzed the reaction mechanism of ba3 oxidase and found evidence that the first proton taken up by the four-electron reduced ba3 oxidase is transferred to a site distant from the catalytic site, the pump site, and that only every second proton taken up from solution is pumped. Data obtained from studies using site-directed mutagenesis and flow-flash spectroscopy suggest a probable location of the pump site.

Under anaerobic conditions, some organisms are able to generate a proton- motive force using nitrate and nitrite as electron acceptors. In this process, the cytotoxic reaction intermediate nitric oxide is produced. Nitric oxide reductase (NOR), a deviant heme-copper oxidase that reduces NO to the rather harmless N2O, does not pump any protons. The catalytic mechanism of nitric oxide reduction by NOR is very poorly understood.

Here we demonstrate that substrate inhibition, which occurs in NOR from Paracoccus denitrificans above 5 μM NO, can already be observed before the electrons from the low-spin hemes re-distribute to the active site. Furthermore, we found that a single specific proton pathway is used for proton-transfer leading from the periplasm to the active site. 

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2015. 64 p.
Keyword
Heme-copper oxidase, electron transfer, proton transfer, nitric oxide reductase, ba3 oxidase, flow-flash, laser-flash photolysis
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-119996 (URN)978-91-7649-263-5 (ISBN)
Public defence
2015-11-23, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2015-10-30 Created: 2015-08-31 Last updated: 2015-10-22Bibliographically approved

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