Low-Spin Heme b3 in the Catalytic Center of Nitric Oxide Reductase from Pseudomonas nautica
| Autor(a) principal: | |
|---|---|
| Data de Publicação: | 2011 |
| Outros Autores: | , , , , , , , |
| Tipo de documento: | Artigo |
| Idioma: | eng |
| Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
| Texto Completo: | http://hdl.handle.net/10362/8618 |
Resumo: | Biochemistry, 2011, 50 (20), pp 4251–4262 DOI: 10.1021/bi101605p |
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Low-Spin Heme b3 in the Catalytic Center of Nitric Oxide Reductase from Pseudomonas nauticaBiochemistry, 2011, 50 (20), pp 4251–4262 DOI: 10.1021/bi101605pRespiratory nitric oxide reductase (NOR) was purified from membrane extract of Pseudomonas (Ps.) nautica cells to homogeneity as judged by polyacrylamide gel electrophoresis. The purified protein is a heterodimer with subunits of molecular masses of 54 and 18 kDa. The gene encoding both subunits was cloned and sequenced. The amino acid sequence shows strong homology with enzymes of the cNOR class. Iron/heme determinations show that one heme c is present in the small subunit (NORC) and that approximately two heme b and one non-heme iron are associated with the large subunit (NORB), in agreement with the available data for enzymes of the cNOR class. Mössbauer characterization of the as-purified, ascorbate-reduced, and dithionite-reduced enzyme confirms the presence of three heme groups (the catalytic heme b(3) and the electron transfer heme b and heme c) and one redox-active non-heme Fe (Fe(B)). Consistent with results obtained for other cNORs, heme c and heme b in Ps. nautica cNOR were found to be low-spin while Fe(B) was found to be high-spin. Unexpectedly, as opposed to the presumed high-spin state for heme b(3), the Mössbauer data demonstrate unambiguously that heme b(3) is, in fact, low-spin in both ferric and ferrous states, suggesting that heme b(3) is six-coordinated regardless of its oxidation state. EPR spectroscopic measurements of the as-purified enzyme show resonances at the g ∼ 6 and g ∼ 2-3 regions very similar to those reported previously for other cNORs. The signals at g = 3.60, 2.99, 2.26, and 1.43 are attributed to the two charge-transfer low-spin ferric heme c and heme b. Previously, resonances at the g ∼ 6 region were assigned to a small quantity of uncoupled high-spin Fe(III) heme b(3). This assignment is now questionable because heme b(3) is low-spin. On the basis of our spectroscopic data, we argue that the g = 6.34 signal is likely arising from a spin-spin coupled binuclear center comprising the low-spin Fe(III) heme b(3) and the high-spin Fe(B)(III). Activity assays performed under various reducing conditions indicate that heme b(3) has to be reduced for the enzyme to be active. But, from an energetic point of view, the formation of a ferrous heme-NO as an initial reaction intermediate for NO reduction is disfavored because heme [FeNO](7) is a stable product. We suspect that the presence of a sixth ligand in the Fe(II)-heme b(3) may weaken its affinity for NO and thus promotes, in the first catalytic step, binding of NO at the Fe(B)(II) site. The function of heme b(3) would then be to orient the Fe(B)-bound NO molecules for the formation of the N-N bond and to provide reducing equivalents for NO reduction.American Chemical SocietyRUNTimóteo, Cristina G.Pereira, Alice S.Martins, Carlos E.Naik, Sunil G.Duarte, Américo G.Moura, José J. G.Tavares, PedroMoura, IsabelHuynh, Boi Hanh2013-01-30T11:38:02Z20112011-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/10362/8618eng0006-2960info:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2024-03-11T03:41:26Zoai:run.unl.pt:10362/8618Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T03:18:19.422180Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
| dc.title.none.fl_str_mv |
Low-Spin Heme b3 in the Catalytic Center of Nitric Oxide Reductase from Pseudomonas nautica |
| title |
Low-Spin Heme b3 in the Catalytic Center of Nitric Oxide Reductase from Pseudomonas nautica |
| spellingShingle |
Low-Spin Heme b3 in the Catalytic Center of Nitric Oxide Reductase from Pseudomonas nautica Timóteo, Cristina G. |
| title_short |
Low-Spin Heme b3 in the Catalytic Center of Nitric Oxide Reductase from Pseudomonas nautica |
| title_full |
Low-Spin Heme b3 in the Catalytic Center of Nitric Oxide Reductase from Pseudomonas nautica |
| title_fullStr |
Low-Spin Heme b3 in the Catalytic Center of Nitric Oxide Reductase from Pseudomonas nautica |
| title_full_unstemmed |
Low-Spin Heme b3 in the Catalytic Center of Nitric Oxide Reductase from Pseudomonas nautica |
| title_sort |
Low-Spin Heme b3 in the Catalytic Center of Nitric Oxide Reductase from Pseudomonas nautica |
| author |
Timóteo, Cristina G. |
| author_facet |
Timóteo, Cristina G. Pereira, Alice S. Martins, Carlos E. Naik, Sunil G. Duarte, Américo G. Moura, José J. G. Tavares, Pedro Moura, Isabel Huynh, Boi Hanh |
| author_role |
author |
| author2 |
Pereira, Alice S. Martins, Carlos E. Naik, Sunil G. Duarte, Américo G. Moura, José J. G. Tavares, Pedro Moura, Isabel Huynh, Boi Hanh |
| author2_role |
author author author author author author author author |
| dc.contributor.none.fl_str_mv |
RUN |
| dc.contributor.author.fl_str_mv |
Timóteo, Cristina G. Pereira, Alice S. Martins, Carlos E. Naik, Sunil G. Duarte, Américo G. Moura, José J. G. Tavares, Pedro Moura, Isabel Huynh, Boi Hanh |
| description |
Biochemistry, 2011, 50 (20), pp 4251–4262 DOI: 10.1021/bi101605p |
| publishDate |
2011 |
| dc.date.none.fl_str_mv |
2011 2011-01-01T00:00:00Z 2013-01-30T11:38:02Z |
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info:eu-repo/semantics/publishedVersion |
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info:eu-repo/semantics/article |
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article |
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publishedVersion |
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http://hdl.handle.net/10362/8618 |
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http://hdl.handle.net/10362/8618 |
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eng |
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eng |
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0006-2960 |
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info:eu-repo/semantics/openAccess |
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openAccess |
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application/pdf |
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American Chemical Society |
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American Chemical Society |
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reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação instacron:RCAAP |
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Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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RCAAP |
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RCAAP |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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