Formation of a cytochrome c-nitrous oxide reductase complex is obligatory for N2O reduction by Paracoccus pantotrophus.

The research article investigates the crucial role of cytochrome c in the reduction of nitrous oxide (N2O) to dinitrogen (N2) by Paracoccus pantotrophus, a denitrifying bacteria. The study explains that active N2OR, a complex with two metal centers including a binuclear copper center (CuA) and a tetranuclear copper-sulfide center (CuZ), remains inactive in the absence of cytochrome c even if there are enough electrons to initiate a turnover.

Objectives and Methodology

• The researchers aimed to understand the workings of nitrous oxide reductase (N2OR), which is responsible for the last step of bacterial denitrification – the two-electron reduction of N2O to N2.
• The experiment involved stopped flow experiments at low ionic strengths to capture the rapid electron transfer between the reduced horse heart (HH) cytochrome c and the CuA center in the fully oxidized N2OR.

Findings

• The research revealed that electron transfer occurs at a fast rate (kobs=150 s-1) when reduced N2OR is mixed with oxidized cytochrome c.
• The internal electron transfer process that occurs subsequently from CuZ to CuA is much slower (kobs=0.1-0.4 s-1), making it the rate-determining step in the catalytic cycle.
• Interestingly, the study notes that in the absence of cytochrome c, fully reduced N2OR doesn’t act on its substrate despite having enough electrons to start a single turnover.

Conclusions and Implications

• The study proposes that a conformational change in N2OR, which is instigated by its specific interaction with cytochrome c, either enables electron transfer between CuA and CuZ or regulates the rate of N2O decomposition at the active site.
• These findings are significant in shedding light on the essential role of cytochrome c in N2O reduction. They emphasize that cytochrome c is not just an electron donor but might also induce important structural changes in N2OR that allow it to function optimally.