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

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 {FeH1NO}(7); ccNiR was not reduced beyond the {FeH1NO}(7) state. The midpoint potentials for sequential reduction of Fe-H1(III)(NO2-) to Fe-H1(II)(NO2-) and then to {FeH1NO}(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 {FeH1NO}(7) is so much easier than the reduction of Fe-H1(III)(NO2-) to Fe-H1(II)(NO2-). With weak reductants, free NO center dot was released from nitrite-loaded ccNiR. The release of NO center dot from {FeH1NO}(7) is exceedingly slow (k similar to 0.001 s(-1)), but it is somewhat faster (k similar to 0.050 s(-1)) while Fe-H1(III)(NO2-) is being reduced to {FeH1NO}(7); then, the release of NO center dot from the undetectable transient {FeH1NO}(6) can compete with reduction of {FeH1NO}(6) to {FeH1NO}(7). CcNiR appears to be optimized to capture nitrite and minimize the release of free NO center dot. Nitrite capture is achieved by reducing bound nitrite with even weak election donpors, while NO center dot release is minimized by stabilizing the substitutionally inert {FeH1NO}(7) over the more labile {FeH1NO}(6).

Automated summary

The decaheme enzyme cytochrome c nitrite reductase facilitates the reduction of nitrite to ammonium with different kinetics of intermediate formation when strong or weak reductants are used, indicating its complex mechanism influenced by the catalyst.