Accommodation of two diatomic molecules in cytochrome bo 3: Insights into NO reductase activity in terminal oxidases

Takahiro Hayashi, Myat T. Lin, Krithika Ganesan, Ying Chen, James A. Fee, Robert B. Gennis, Pierre Moenne-Loccoz

Research output: Contribution to journalArticle

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Abstract

Bacterial heme-copper terminal oxidases react quickly with NO to form a heme - nitrosyl complex, which, in some of these enzymes, can further react with a second NO molecule to produce N 2O. Previously, we characterized the heme a 3-NO complex formed in cytochrome ba 3 from Thermus thermophilus and the product of its low-temperature illumination. We showed that the photolyzed NO group binds to Cu B(I) to form an end-on NO-Cu B or a side-on copper-nitrosyl complex, which is likely to represent the binding characteristics of the second NO molecule at the heme-copper active site. Here we present a comparative study with cytochrome bo 3 from Escherichia coli. Both terminal oxidases are shown to catalyze the same two-electron reduction of NO to N 2O. The EPR and resonance Raman signatures of the heme o 3-NO complex are comparable to those of the a 3-NO complex. However, low-temperature FTIR experiments reveal that photolysis of the heme o 3-NO complex does not produce a Cu B-nitrosyl complex, but that instead, the NO remains unbound in the active-site cavity. Additional FTIR photolysis experiments on the heme-nitrosyl complexes of these terminal oxidases, in the presence of CO, demonstrate that an [o 3-NO·OC-Cu B] tertiary complex can form in bo 3 but not in ba 3. We assign these differences to a greater iron-copper distance in the reduced form of bo 3 compared to that of ba 3. Because this difference in metal-metal distance does not appear to affect the NO reductase activity, our results suggest that the coordination of the second NO to Cu B is not an essential step of the reaction mechanism.

Original languageEnglish (US)
Pages (from-to)883-890
Number of pages8
JournalBiochemistry
Volume48
Issue number5
DOIs
StatePublished - Feb 10 2009

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Cytochromes
Heme
Oxidoreductases
Molecules
Copper
Photolysis
Fourier Transform Infrared Spectroscopy
Catalytic Domain
Metals
Thermus thermophilus
Temperature
Carbon Monoxide
Lighting
Escherichia coli
Paramagnetic resonance
Iron
Experiments
Electrons
Enzymes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Accommodation of two diatomic molecules in cytochrome bo 3 : Insights into NO reductase activity in terminal oxidases. / Hayashi, Takahiro; Lin, Myat T.; Ganesan, Krithika; Chen, Ying; Fee, James A.; Gennis, Robert B.; Moenne-Loccoz, Pierre.

In: Biochemistry, Vol. 48, No. 5, 10.02.2009, p. 883-890.

Research output: Contribution to journalArticle

Hayashi, Takahiro ; Lin, Myat T. ; Ganesan, Krithika ; Chen, Ying ; Fee, James A. ; Gennis, Robert B. ; Moenne-Loccoz, Pierre. / Accommodation of two diatomic molecules in cytochrome bo 3 : Insights into NO reductase activity in terminal oxidases. In: Biochemistry. 2009 ; Vol. 48, No. 5. pp. 883-890.
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AU - Fee, James A.

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N2 - Bacterial heme-copper terminal oxidases react quickly with NO to form a heme - nitrosyl complex, which, in some of these enzymes, can further react with a second NO molecule to produce N 2O. Previously, we characterized the heme a 3-NO complex formed in cytochrome ba 3 from Thermus thermophilus and the product of its low-temperature illumination. We showed that the photolyzed NO group binds to Cu B(I) to form an end-on NO-Cu B or a side-on copper-nitrosyl complex, which is likely to represent the binding characteristics of the second NO molecule at the heme-copper active site. Here we present a comparative study with cytochrome bo 3 from Escherichia coli. Both terminal oxidases are shown to catalyze the same two-electron reduction of NO to N 2O. The EPR and resonance Raman signatures of the heme o 3-NO complex are comparable to those of the a 3-NO complex. However, low-temperature FTIR experiments reveal that photolysis of the heme o 3-NO complex does not produce a Cu B-nitrosyl complex, but that instead, the NO remains unbound in the active-site cavity. Additional FTIR photolysis experiments on the heme-nitrosyl complexes of these terminal oxidases, in the presence of CO, demonstrate that an [o 3-NO·OC-Cu B] tertiary complex can form in bo 3 but not in ba 3. We assign these differences to a greater iron-copper distance in the reduced form of bo 3 compared to that of ba 3. Because this difference in metal-metal distance does not appear to affect the NO reductase activity, our results suggest that the coordination of the second NO to Cu B is not an essential step of the reaction mechanism.

AB - Bacterial heme-copper terminal oxidases react quickly with NO to form a heme - nitrosyl complex, which, in some of these enzymes, can further react with a second NO molecule to produce N 2O. Previously, we characterized the heme a 3-NO complex formed in cytochrome ba 3 from Thermus thermophilus and the product of its low-temperature illumination. We showed that the photolyzed NO group binds to Cu B(I) to form an end-on NO-Cu B or a side-on copper-nitrosyl complex, which is likely to represent the binding characteristics of the second NO molecule at the heme-copper active site. Here we present a comparative study with cytochrome bo 3 from Escherichia coli. Both terminal oxidases are shown to catalyze the same two-electron reduction of NO to N 2O. The EPR and resonance Raman signatures of the heme o 3-NO complex are comparable to those of the a 3-NO complex. However, low-temperature FTIR experiments reveal that photolysis of the heme o 3-NO complex does not produce a Cu B-nitrosyl complex, but that instead, the NO remains unbound in the active-site cavity. Additional FTIR photolysis experiments on the heme-nitrosyl complexes of these terminal oxidases, in the presence of CO, demonstrate that an [o 3-NO·OC-Cu B] tertiary complex can form in bo 3 but not in ba 3. We assign these differences to a greater iron-copper distance in the reduced form of bo 3 compared to that of ba 3. Because this difference in metal-metal distance does not appear to affect the NO reductase activity, our results suggest that the coordination of the second NO to Cu B is not an essential step of the reaction mechanism.

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