Activation of the Cytochrome cd1 Nitrite Reductase from Paracoccus pantotrophus

Abstract

Cytochromes cd 1 are dimeric bacterial nitrite reductases, which contain two hemes per monomer. On reduction of both hemes, the distal ligand of heme d 1 dissociates, creating a vacant coordination site accessible to substrate. Heme c, which transfers electrons from donor proteins into the active site, has histidine/methionine ligands except in the oxidized enzyme from Paracoccus pantotrophus where both ligands are histidine. During reduction of this enzyme, Tyr 25 dissociates from the distal side of heme d 1 , and one heme c ligand is replaced by methionine. Activity is associated with histidine/methionine coordination at heme c, and it is believed that P. pantotrophus cytochrome cd 1 is unreactive toward substrate without reductive activation. However, we report here that the oxidized enzyme will react with nitrite to yield a novel species in which heme d 1 is EPR-silent. Magnetic circular dichroism studies indicate that heme d 1 is low-spin Fe III but EPR-silent as a result of spin coupling to a radical species formed during the reaction with nitrite. This reaction drives the switch to histidine/methionine ligation at Fe III heme c. Thus the enzyme is activated by exposure to its physiological substrate without the necessity of passing through the reduced state. This reactivity toward nitrite is also observed for oxidized cytochrome cd 1 from Pseudomonas stutzeri suggesting a more general involvement of the EPR-silent Fe III heme d 1 species in nitrite reduction.

Modulation of the Ligand-Field Anisotropy in a Series of Ferric Low-Spin Cytochrome c Mutants derived from Pseudomonas aeruginosa Cytochrome c-551 and Nitrosomonas europaea Cytochrome c-552: A Nuclear Magnetic Resonance and Electron Paramagnetic Resonance Study

“…The correlation shows that i) when the g max signal increases, the average methyl chemical shift <δ> increases and ii) when the ligand field anisotropy V/Δ increases (from more axial to rhombic), the average methyl chemical shift <δ> decreases. The linear correlation holds fairly well when other experimental data ( g max , V/Δ and <δ>) available for similar cytochrome c proteins with His/Met axial ligation are included ( g max f (<δ>) with R = 0.93, V/Δ f (<δ>) with R = 0.95) such as horse cytochrome c 61, P. stutzeri c -551,47,65 P. ZoBell c -551,47,66 Rhodopseudomonas palustris c -2,67 and Saccharomyces cerevisiae iso-1 cyt c 47,68. However, the linear trend observed does not accommodate Bacillus pasteurii c -55369 and Rhodospirillum rubrum c -268, for which g max , V/Δ and <δ> values fall well outside the linear region.…”

Very Early Reaction Intermediates Detected by Microsecond Time Scale Kinetics of Cytochrome cd1-catalyzed Reduction of Nitrite

“…No peak between 550 and 630 nm was seen for the Y25S enzyme with either nitrite or NO bound, further indicating that the 620 nm peak does not arise from either of these ligands bound to ferric d 1 heme. Recent work has invoked the possibility of electron donation from an amino acid side chain during nitrite reduction by cytochrome cd 1 (20), and therefore formation of cFe(II)⅐d 1 Fe(II)-NO was also addressed. However, when the 130-s sample was subjected to EPR, no signal arising from Fe(II)-NO was observed.…”

Formation of Nitric Oxide from Nitrite by the Ferriheme b Protein Nitrophorin 7

“…19 The disproportionation of NO 2 -readily occurs in strongly acidic solution, but the rate is extremely low at pH g7. 20 Although the reaction is not promoted by heme proteins, 3 it was observed in a few model ferrihemes with exclusion of water.…”