A Kinetic Investigation of the Early Steps in Cytochrome c Nitrite Reductase (ccNiR)-Catalyzed Reduction of Nitrite

Abstract

The decaheme enzyme cytochrome c nitrite reductase (ccNiR) catalyzes reduction of nitrite to ammonium in a six-electron, eight-proton process. With a strong reductant as the electron source, ammonium is the sole product. However, intermediates accumulate when weaker reductants are employed, facilitating study of the ccNiR mechanism. Herein, the early stages of Shewanella oneidensis ccNiR-catalyzed nitrite reduction were investigated by using the weak reductants N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) and ferrocyanide. In stopped-flow experiments, reduction of nitrite-loaded ccNiR by TMPD generated a transient intermediate, identified as Fe_H1^II(NO₂^-), where Fe_H1 represents the ccNiR active site. Fe_H1^II(NO₂^-) accumulated rapidly and was then more slowly converted to the two-electron-reduced moiety {Fe_H1NO}⁷; ccNiR was not reduced beyond the {Fe_H1NO}⁷ state. The midpoint potentials for sequential reduction of Fe_H1^III(NO₂^-) to Fe_H1^II(NO₂^-) and then to {Fe_H1NO}⁷ were estimated to be 130 and 370 mV versus the standard hydrogen electrode, respectively. Fe_H1^II(NO₂^-) does not accumulate at equilibrium because its reduction to {Fe_H1NO}⁷ is so much easier than the reduction of Fe_H1^III(NO₂^-) to Fe_H1^II(NO₂^-). With weak reductants, free NO• was released from nitrite-loaded ccNiR. The release of NO• from {Fe_H1NO}⁷ is exceedingly slow (k ∼ 0.001 s^-1), but it is somewhat faster (k ∼ 0.050 s^-1) while Fe_H1^III(NO₂^-) is being reduced to {Fe_H1NO}⁷; then, the release of NO• from the undetectable transient {Fe_H1NO}⁶ can compete with reduction of {Fe_H1NO}⁶ to {Fe_H1NO}⁷. CcNiR appears to be optimized to capture nitrite and minimize the release of free NO•. Nitrite capture is achieved by reducing bound nitrite with even weak electron donors, while NO• release is minimized by stabilizing the substitutionally inert {Fe_H1NO}⁷ over the more labile {Fe_H1NO}⁶.