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

Abstract

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.

Characterization of the nitric oxide reductase from Thermus thermophilus

“…Therefore, assays were performed at 42°C, which is below the optimum growth temperature. The turnover number (k cat ) obtained at 42°C for the TtcNOR is considerably smaller than those reported for other cNORs (5,6,13,16,18,20). The highest activity obtained with the TtcNOR was about 9 electrons (e − )/min (at 42°C) with the electron donor PMS (phenazine methosulfate) ( Table 1), which presumably donates electrons directly to the active site in the NorB subunit without involving the heme c in the NorC subunit (5).…”

“…Enzymes belonging to the cNOR family have been isolated from five denitrifying bacteria: Pseudomonas stutzeri (12, 13), Paracoccus denitrificans (14,15), Halomonas halodenitrificans (16,17), Roseobacter denitrificans (18,19), Pseudomonas nautica (20), and Pseudomonas aeruginosa, for which an X-ray structure has been reported (5,21). All of these strains are in the Proteobacteria phylum.…”

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

“…NORs have been isolated from different denitrifying organisms, such as Paracoccus denitrificans [1,2], and several Pseudomonas species, such as stutzeri [3,4], aeruginosa [5] and nautica [6], currently known as Marinobacter hydrocarbonoclasticus [7]. This enzyme belongs to the bacterial denitrification pathway (NO 3 − → NO 2 − → NO → N 2 O → N 2 ), where NO 3 − is the electron acceptor, being reduced to N 2 in consecutive redox reactions, mediated by different metalloenzymes [8,9].…”

Crystal structures of nitric oxide reductases provide key insights into functional conversion of respiratory enzymes

“…There are three proposed mechanisms on the basis of the structural, spectroscopic, and theoretical studies (26)(27)(28)(29). Using a rapid freeze quench method combined with electron paramagnetic resonance spectroscopy, we found that simultaneous formation of ferrous heme-NO and ferrous Fe B -NO at 0.5 ms after the introduction of NO into fully reduced cNOR (26).…”

“…In cis-heme b 3 mechanism, first NO molecule coordinates to heme b 3 , followed by the cis-hyponitrite formation upon the direct binding of second NO to heme-bound NO. There is also alternative suggestion of cis-Fe B mechanism in which two NO molecules bind to nonheme Fe B (29), because ferrous heme-NO species would be highly stable dead end product.…”