The effect of haem ligands on the redox states of the hexa-haem nitrite reductase from Wolinella succinogenes

Abstract

The nitrite reductase of Wolinella succinogenes containing six covalently bound haem groups has one haem group that will not reduce fully in the presence of excess Na2S2O4. The effect of the extrinsic ligands CO and cyanide on the redox state of this haem was studied by e.p.r. and magnetic c.d. spectroscopy. It was found that both ligands increased the extent of reduction of this haem group, and that in the case of CO binding the level of reduction was correlated with the extent of CO saturation of the enzyme. Stopped-flow studies of the effect of cyanide binding on the rate of dithionite reduction showed that the rate of reduction of the ligand-binding site was increased in the presence of cyanide. This suggests that reduction of the haem groups at the active site is thermodynamically unfavourable in the absence of an extrinsic ligand. The role of the 'non-reducing' haem group and the effect of ligands on this centre and on the rate of reduction are discussed in relation to the reduction of nitrite by this enzyme.

X-ray Crystallographic Study of Cyanide Binding Provides Insights into the Structure-Function Relationship for Cytochromecd 1 Nitrite Reductase from Paracoccus pantotrophus

“…The active-site heme is also surrounded by amino acid residues that are likely to be protonated, contributing to a positive electrostatic potential when nitrite binds (33). Strong cyanide binding to the reduced form of this type of enzyme from Wollinella succinogenes has been reported (34). We argue that the lysine in the heme binding motif is of crucial importance in promoting the binding of anions, and the environment of this heme contributes to the provision of a reduction potential low enough to reduce substrate nitrite through to ammonia.…”

An analysis of the reaction kinetics of the hexahaem nitrite reductase of the anaerobic rumen bacterium Wolinella succinogenes

“…In the resting form a marked band at 610 nm is seen in the enzyme's absorption spectrum, indicative of high-spin ferric haem, whereas the redoxcycled form is characterized by the presence of a broader ferric Soret band. There is no evidence that corresponding resting and redox-cycled forms of the enzyme exist in the reduced enzyme (Blackmore et al, 1990a).…”

“…The redox-cycled form also exhibits two kinetic steps, but, unlike the resting form, the rate of the slower of these steps shows a linear dithionite-concentration-dependence (Blackmore et al, 1990b). It has also been found that the low rate of dithionite reduction of resting nitrite reductase increased in the presence of cyanide, but the rate limit of this process was unchanged (Blackmore et al, 1990a). This can be accounted for if the reduction of the active-site ligand-binding haems is unfavourable owing to the low redox potential of the exchangecoupled haem pair.…”