Elucidating Electron Storage and Distribution within the Pentaheme Scaffold of Cytochrome c Nitrite Reductase (NrfA)

Alfaro, Victor Sosa; Campeciño, Julius; Tracy, Matthew; Elliott, Sean J.; Hegg, Eric L.; Lehnert, Nicolai

doi:10.1021/acs.biochem.0c00977

Elucidating Electron Storage and Distribution within the Pentaheme Scaffold of Cytochrome c Nitrite Reductase (NrfA)

Journal Article · Tue Jun 01 00:00:00 EDT 2021 · Biochemistry

DOI:https://doi.org/10.1021/acs.biochem.0c00977· OSTI ID:1865050

^[1]; Campeciño, Julius ^[2]; Tracy, Matthew ^[3]; ^[3]; ^[2]; ^[4]

Univ. of Michigan, Ann Arbor, MI (United States); University of Michigan
Michigan State Univ., East Lansing, MI (United States)
Boston Univ., MA (United States)
Univ. of Michigan, Ann Arbor, MI (United States)

Cytochrome c nitrite reductases (CcNIR or NrfA) play important roles in the global nitrogen cycle by conserving the usable nitrogen in the soil. Here, the electron storage and distribution properties within the pentaheme scaffold of Geobacter lovleyi NrfA were investigated via EPR spectroscopy coupled with chemical titration experiments. Initially, a chemical reduction method was established to sequentially add electrons to the fully oxidized protein, one equivalent at a time. The step–by–step reduction of the hemes was then followed using UV–Vis and EPR spectroscopy. EPR–spectral simulations elucidate the sequence of heme reduction within the pentaheme scaffold, and identify the signals of all five hemes in the EPR spectra. Electrochemical experiments ascertain the reduction potentials for each heme, observed in a narrow +11 (Heme 5) to –212 mV (Heme 3) range (vs SHE). Based on quantitative analysis/simulation of the EPR data, we demonstrate that Hemes 4 and 5 are reduced first (before the active site Heme 1) and serve the purpose of an electron storage unit within the protein. To probe the role of the central Heme 3, a H108M NrfA variant was generated where the reduction potential of Heme 3 is shifted positively (from –212 mV to +29 mV). Here, the H108M mutation significantly impacts the distribution of electrons within the pentaheme scaffold and the reduction potentials of the hemes, reducing the catalytic activity of the enzyme to 3% compared to wild–type. We propose that this is due to Heme 3’s important role as an electron gateway in the wild–type enzyme.

View Accepted Manuscript (DOE)

Research Organization:: Univ. of Michigan, Ann Arbor, MI (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES)

Grant/Contract Number:: SC0018173; SC0017952

OSTI ID:: 1865050

Alternate ID(s):: OSTI ID: 1852451

Journal Information:: Biochemistry, Journal Name: Biochemistry Journal Issue: 23 Vol. 60; ISSN 0006-2960

Publisher:: American Chemical Society (ACS)Copyright Statement

Country of Publication:: United States

Language:: English

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Elucidating Electron Storage and Distribution within the Pentaheme Scaffold of Cytochrome c Nitrite Reductase (NrfA)

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