Ultrafast ligand binding dynamics in the active site of native bacterial nitric oxide reductase

Abstract

The active site of nitric oxide reductase from Paracoccus denitrificans contains heme and non-heme iron and is evolutionarily related to heme-copper oxidases. The CO and NO dynamics in the active site were investigated using ultrafast transient absorption spectroscopy. We find that, upon photodissociation from the active site heme, 20% of the CO rebinds in 170 ps, suggesting that not all the CO transiently binds to the non-heme iron. The remaining 80% does not rebind within 4 ns and likely migrates out of the active site without transient binding to the non-heme iron. Rebinding of NO to ferrous heme takes place in approximately 13 ps. Our results reveal that heme-ligand recombination in this enzyme is considerably faster than in heme-copper oxidases and are consistent with a more confined configuration of the active site.

The bacterial respiratory nitric oxide reductase

“…After photolysis, the apparent bimolecular rate of CO rebinding to the haem a 3 in cytochrome aa 3 is limited by transient binding to Cu B , but in NOR, CO recombination is apparently diffusion limited (k on = 1.7 × 10 8 M −1 ·s −1 ) [31,52,53], inferring that Fe B does not act as an intermediate binding site. This is consistent with an ultrafast study of CO recombination to fully reduced NOR, which indicated a significant phase (20%) of geminate recombination and also found no evidence for interaction with Fe B [49].…”

“…It is, however, known that NO will react with the fully oxidized enzyme [49,50], and the immediate product of this reaction has been characterized by resonance Raman spectroscopy as 6C-ferric nitrosyl haem b 3 [50]. In time, and perhaps depending on experimental conditions, a mixture of ferric and ferrous haem b 3 -NO complexes is obtained [49]. This surprising observation can be explained in terms of the binding of NO favouring the ferrous state of haem b 3 , raising the effective reduction potential of the haem b 3 and trapping any electrons, possibly derived from the buffer [51], at that site.…”

“…This is particularly interesting given the low reduction potential reported for haem b 3 [37] and the similarly low reduction potential of Fe B [9], which implies that it might be difficult for high-potential electron donors such as pseudoazurin to fully reduce the dinuclear centre. It is, however, known that NO will react with the fully oxidized enzyme [49,50], and the immediate product of this reaction has been characterized by resonance Raman spectroscopy as 6C-ferric nitrosyl haem b 3 [50]. In time, and perhaps depending on experimental conditions, a mixture of ferric and ferrous haem b 3 -NO complexes is obtained [49].…”

(Un)suitability of the use of pH buffers in biological, biochemical and environmental studies and their interaction with metal ions – a review

“…Borges and Collins 112 described that buffers, such as tricine (pH 8.0, 20 mmol l À1 ), affect the high-performance liquid chromatography (HPLC) stability and performance of study have themselves high complexing capabilities and buffer interference is thought to be simply nonexistent. These studies involve proteins with heme groups, [134][135][136] Zn-nger motifs, [137][138][139] metalloproteins 116,133,[140][141][142] and/or other complexing agents in solution. 81,[143][144][145] In fact, the concentrations of the compounds used, and most importantly the ratio of the buffer concentration to the complexing compound concentration in the medium, are within values that support the idea that no interference of the buffer occurs.…”

Roles of glutamates and metal ions in a rationally designed nitric oxide reductase based on myoglobin

“…Some members of HCOs such as cytochrome cbb 3 oxidase display NOR activity (26-28), although the activity is ∼50-fold lower than native NOR (26). Therefore, it is important to elucidate the structural features, specifically the roles of the conserved glutamates close to the Fe B site and metal ions (copper vs. iron), responsible for the reduction of NO to N 2 O.To address these issues, biochemical and biophysical studies of native NOR and its variants have been carried out (24,25,(29)(30)(31)(32)(33)(34)(35)(36)(37). For example, Richardson and coworkers investigated the effects of amino acid substitutions of the five conserved glutamates (E122 and E125 presumed to face the periplasm and E198, E202, and E267 located in the interior of the membrane, close to the catalytic site) in the catalytic subunit of Paracoccus denitrificans, NorB.…”