Substrate Control of Internal Electron Transfer in Bacterial Nitric-oxide Reductase

Lachmann, Peter; Huang, Yafei; Reimann, Joachim; Flock, Ulrika; Ädelroth, Pia

doi:10.1074/jbc.m110.123984

Cited by 31 publications

(57 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Without further kinetic data, we cannot exclude that at a later mechanistic step heme b 3 will somehow bind to NO (or to a reaction intermediate). Recently, Lachmann and coworkers (52) demonstrated that CO binds to reduced heme b 3 (probably displacing the sixth ligand initially present) and that flash-induced dissociation causes heme b 3 oxidation and catalysis. It is also known that for the CO-bound enzyme, NO can still bind to the Fe B site (14).…”

Section: Discussionmentioning

confidence: 99%

Low-Spin Heme b₃ in the Catalytic Center of Nitric Oxide Reductase from Pseudomonas nautica

et al. 2011

View full text Add to dashboard Cite

Respiratory 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 b3, and the electron transfer heme b and heme c) and one redox-active non-heme Fe (FeB). Consistent with results obtained for other cNORs, heme c and heme b in Ps. nautica cNOR were found to be low-spin while FeB was found to be high-spin. Unexpectedly, as opposed to the presumed high-spin state for heme b3, the Mössbauer data demonstrate unambiguously that heme b3 is, in fact, low-spin in both ferric and ferrous states, suggesting that heme b3 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 FeIII heme b3. This assignment is now questionable because heme b3 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 FeIII heme b3 and the high-spin FeBIII. Activity assays performed under various reducing conditions indicate that heme b3 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 FeII-heme b3 may weaken its affinity for NO and thus promotes, in the first catalytic step, binding of NO at the FeBII site. The function of heme b3 would then be, to orient the FeB-bound NO molecules for the formation of the N-N bound and to provide reducing equivalents for NO reduction.

show abstract

Section: Discussionmentioning

confidence: 99%

Low-Spin Heme b₃ in the Catalytic Center of Nitric Oxide Reductase from Pseudomonas nautica

et al. 2011

View full text Add to dashboard Cite

show abstract

“… and Ref. on a set‐up custom built by Applied Photophysics (Leatherhead, UK). Briefly, samples of ~ 5 μ m c NOR (in 50 m m HEPES, pH 7.5, 50 m m KCl, 0.05% DDM, 0.2 μ m PMS, 30 m m glucose, 20 U·mL −1 catalase) were prepared in modified Thunberg cuvette.…”

Section: Methodsmentioning

confidence: 99%

Insights into the mechanism of nitric oxide reductase from a Fe_B‐depleted variant

et al. 2019

Self Cite

View full text Add to dashboard Cite

A key step of denitrification, the reduction of toxic nitric oxide to nitrous oxide, is catalysed by cytochrome c‐dependent NO reductase (cNOR). cNOR contains four redox‐active cofactors: three hemes and a nonheme iron (FeB). Heme b3 and FeB constitute the active site, but the specific mechanism of NO‐binding events and reduction is under debate. Here, we used a recently constructed, fully folded and hemylated cNOR variant that lacks FeB to investigate the role of FeB during catalysis. We show that in the FeB‐less cNOR, binding of both NO and O2 to heme b3 still occurs but further reduction is impaired, although to a lesser degree for O2 than for NO. Implications for the catalytic mechanisms of cNOR are discussed.

show abstract

“…73 s −1 [37]). Lachmann and co-workers [39] used spectroscopic methods to determined re-oxidation rate constants of the NOR metal co-factors under NO turnover, however, the determined values present substrate concentration dependence and are lower (c.a. 12 s −1 [39]) than the ones determined for the direct ET between the enzyme and the graphite electrode [37].…”

Section: The No Inhibitory Profilementioning

confidence: 97%

“…This hypothesis was strongly supported by the stability of heme Fe III -nitrosyl compounds [11]. Lachmann and co-workers used flow-flash measurements in single turnover conditions demonstrating that substrate concentration controlled the intramolecular electron transfer (ET) in the NO reduction mechanism and the substrate binding to the inhibition site occurs before the electron redistribution to the catalytic centre [39]. Recently, a theoretical study developed with DFT calculations has indicated a weak interaction between NO and the oxidised binuclear iron centre, suggesting an inhibitory profile caused by nitrate formation from a side reaction [28].…”

Section: Introductionmentioning

confidence: 93%

Steady-state kinetics with nitric oxide reductase (NOR): New considerations on substrate inhibition profile and catalytic mechanism

Duarte

Cordas

Moura

et al. 2014

Biochimica et Biophysica Acta (BBA) - Bioenergetics

View full text Add to dashboard Cite

Nitric oxide reductase (NOR) from denitrifying bacteria is an integral membrane protein that catalyses the two electron reduction of NO to N2O, as part of the denitrification process, being responsible for an exclusive reaction, the NN bond formation, the key step of this metabolic pathway. Additionally, this class of enzymes also presents residual oxidoreductase activity, reducing O2 to H2O in a four electron/proton reaction. In this work we report, for the first time, steady-state kinetics with the Pseudomonas nautica NOR, either in the presence of its physiological electron donor (cyt. c552) or immobilised on a graphite electrode surface, in the presence of its known substrates, namely NO or O2. The obtained results show that the enzyme has high affinity for its natural substrate, NO, and different kinetic profiles according to the electron donor used. The kinetic data, as shown by the pH dependence, is modelled by ionisable amino acid residues nearby the di-nuclear catalytic site. The catalytic mechanism is revised and a mononitrosyl-non-heme Fe complex (FeB(II)-NO) species is favoured as the first catalytic intermediate involved on the NO reduction.

show abstract

Substrate Control of Internal Electron Transfer in Bacterial Nitric-oxide Reductase

Cited by 31 publications

References 35 publications

Low-Spin Heme b₃ in the Catalytic Center of Nitric Oxide Reductase from Pseudomonas nautica

Low-Spin Heme b₃ in the Catalytic Center of Nitric Oxide Reductase from Pseudomonas nautica

Insights into the mechanism of nitric oxide reductase from a Fe_B‐depleted variant

Steady-state kinetics with nitric oxide reductase (NOR): New considerations on substrate inhibition profile and catalytic mechanism

Contact Info

Product

Resources

About

Substrate Control of Internal Electron Transfer in Bacterial Nitric-oxide Reductase

Cited by 31 publications

References 35 publications

Low-Spin Heme b3 in the Catalytic Center of Nitric Oxide Reductase from Pseudomonas nautica

Low-Spin Heme b3 in the Catalytic Center of Nitric Oxide Reductase from Pseudomonas nautica

Insights into the mechanism of nitric oxide reductase from a FeB‐depleted variant

Steady-state kinetics with nitric oxide reductase (NOR): New considerations on substrate inhibition profile and catalytic mechanism

Contact Info

Product

Resources

About

Low-Spin Heme b₃ in the Catalytic Center of Nitric Oxide Reductase from Pseudomonas nautica

Low-Spin Heme b₃ in the Catalytic Center of Nitric Oxide Reductase from Pseudomonas nautica

Insights into the mechanism of nitric oxide reductase from a Fe_B‐depleted variant