Poster Presentation 21st International Conference on Biological Inorganic Chemistry 2025

Constructing a synthetic cytochrome c nitrite reductase microcompartment to analyze the effects of biomimetic confinement on metalloenzyme catalysis (#476)

Joel F Landa 1 , Kyleigh L Range 1 2 , Timothy K Chiang 2 , Arinita Pramanik 2 , Corie Y Ralston 2 , Cheryl A Kerfeld 1 2 , Eric L Hegg 1
  1. Michigan State University, East Lansing, MICHIGAN, United States
  2. Molecular Foundry Division, Lawrence Berkeley National Laboratory, Berkeley, CA, United States

Bacterial Microcompartments (BMC) are proteinaceous organelles made of self-assembling protein subunits that allow bacteria to co-localize enzymatic pathways and sequester toxic intermediates (1). BMCs offer a diverse platform for fine tuning catalysis. NrfA is a penta-heme cytochrome c nitrite reductase that catalyzes the incredible 6-electron reduction of nitrite to the energy rich product ammonium (2). NrfA matures in the periplasm physically separated from cytosolic BMC assembly (3). Therefore, there is need for in vitro assembly methods to encapsulate non-native cargo that are incompatible with in vivo encapsulation. The aim of this research is to construct a synthetic NrfA BMC to study the effects of biomimetic confinement on catalysis. To achieve this, the Center for Catalysis in Biomimetic Confinement has employed a novel chaotropic assembly method to encapsulate NrfA in a synthetic BMC system derived from the organism Haliangium ochraceum (HO) (4). A colorimetric activity assay was used to measure NrfA turnover at different substrate concentrations. NrfA was successfully encapsulated and retained activity with a Vmax = 0.50 ± 0.04 uM/s and a KM = 49.4 ± 25.5. These results indicate that chaotropic in vitro assembly is a viable method for encapsulating large, non-native, active, enzymatic cargo in the HO BMC system. The KM of this system was higher than previously reported for NrfA, indicating that encapsulation may hinder substrate access. Future experiments a planned to optimize the loading efficiency for more accurate kinetic measurements, determine the Michaelis-Menten parameters for distinct configurations of the HO BMC, and determine if encapsulation impacts enzyme stability under stress conditions. This research brings us closer to understanding the effects of encapsulating important metalloenzyme catalysts, and it broadens the applications of BMCs for the biosynthesis of important molecules like ammonium.

 

  1. 1. Chowdhury, C.; Sinha, S.; Chun, S.; Yeates, T. O.; Bobik, T. A. Diverse Bacterial Microcompartment Organelles. Microbiol Mol Biol Rev 2014, 78 (3), 438–468. https://doi.org/10.1128/MMBR.00009-14.
  2. 2. Einsle, O. Cytochrome c Nitrite Reductase. In Encyclopedia of Inorganic and Bioinorganic Chemistry; Scott, R. A., Ed.; Wiley, 2004. https://doi.org/10.1002/9781119951438.eibc0558.
  3. 3. Simon, J.; Gross, R.; Einsle, O.; Kroneck, P. M. H.; Kröger, A.; Klimmek, O. A NapC/NirT-Type Cytochrome c (NrfH) Is the Mediator between the Quinone Pool and the Cytochrome c Nitrite Reductase of Wolinella Succinogenes: Cytochrome c Nitrite Reductase of Wolinella Succinogenes. Molecular Microbiology 2002, 35 (3), 686–696. https://doi.org/10.1046/j.1365-2958.2000.01742.x.
  4. 4. Range, K. L.; Chiang, T. K.; Pramanik, A.; Landa, J. F.; Snyder, S. N.; Zuo, X.; Tiede, D. M.; Utschig, L. M.; Hegg, E. L.; Sutter, M.; Kerfeld, C. A.; Ralston, C. Y. Chaotrope-Based Approach for Rapid In Vitro Assembly and Loading of Bacterial Microcompartment Shells. ACS Nano 2025, acsnano.4c15538. https://doi.org/10.1021/acsnano.4c15538.