Nitrite reductase activity within an antiparallel de novo scaffold

Area of Science:

• Biochemistry
• Bioinorganic Chemistry
• Protein Design

Background:

• Copper nitrite reductase (CuNiR) is crucial for the bacterial nitrogen cycle, converting nitrite to nitric oxide.
• The CuHis3 active site is a key target for modeling and de novo protein design.
• Previous work established symmetric CuNiR models in parallel coiled coils with varying activities.

Purpose of the Study:

• To investigate the CuHis3 binding site within an antiparallel helical bundle scaffold for asymmetric constructs.
• To compare the activity of CuNiR models in antiparallel versus parallel scaffolds.
• To explore structure-activity relationships in de novo designed nitrite reductases.

Main Methods:

• Design and construction of asymmetric CuHis3 binding sites in an antiparallel three-helix bundle.
• Activity assays to measure nitrite reductase function.
• Copper(I) X-ray absorption spectroscopy to analyze electronic transitions.

Main Results:

• A simple CuHis3 site in the antiparallel scaffold showed enhanced activity over parallel constructs.
• More complex designs or repositioning of the binding site reduced activity significantly (up to 15-fold).
• A blue shift in the 1s to 4p transition energy correlated with enhanced activity in imidazole-based constructs.

Conclusions:

• The antiparallel scaffold facilitates the design of active CuNiR models with tunable activity.
• Structure-activity correlations, including spectroscopic data, provide insights for optimizing enzyme design.
• This work progresses towards creating fully functional de novo designed nitrite reductases.