Abstract

NADPH-dependent assimilatory sulfite reductase (SiR) from Escherichia coli performs a six-electron reduction of sulfite to the bioavailable sulfide. SiR is composed of a flavoprotein (SiRFP) reductase subunit and a hemoprotein (SiRHP) oxidase subunit. There is no known high-resolution structure of SiR or SiRFP, thus we do not yet fully understand how the subunits interact to perform their chemistry. Here, we used small-angle neutron scattering to understand the impact of conformationally restricting the highly mobile SiRFP octamer into an electron accepting (closed) or electron donating (open) conformation, showing that SiR remains active, flexible, and asymmetric even with these conformational restrictions. From these scattering data, we then model the first solution structure of SiRFP. Further, computational modeling of the N-terminal 52 amino acids that are responsible for SiRFP oligomerization suggests an eight-helical bundle tethers together the SiRFP subunits to form the SiR core. Finally, mass spectrometry analysis of the closed SiRFP variant show that SiRFP is capable of inter-molecular domain crossover, in which the electron donating domain from one polypeptide is able to interact directly with the electron accepting domain of another polypeptide. This structural characterization suggests that SiR performs its high-volume electron transfer through both inter- and intramolecular pathways between SiRFPmore » domains and, thus, cis or trans transfer from reductase to oxidase subunits. Such highly redundant potential for electron transfer makes this system a potential target for designing synthetic enzymes.« less

Authors:

Walia, Nidhi  ^[1];

• Search DOE PAGES for author "Walia, Nidhi"
• Search DOE PAGES for ORCID "0000-0002-6432-9433"

Murray, Daniel T. ^[2]; Garg, Yashika  ^[1];

• Search DOE PAGES for author "Garg, Yashika"
• Search DOE PAGES for ORCID "0000-0002-4866-3079"

He, Huan  ^[3];

• Search DOE PAGES for author "He, Huan"
• Search DOE PAGES for ORCID "0000-0002-5014-9124"

Weiss, Kevin L.  ^[4];

• Search DOE PAGES for author "Weiss, Kevin L."
• Search DOE PAGES for ORCID "0000-0002-6486-8007"

Nagy, Gergely  ^[4];

• Search DOE PAGES for author "Nagy, Gergely"
• Search DOE PAGES for ORCID "0000-0003-2742-0198"

Elizabeth Stroupe, M. ^[1]

---

+ Show Author Affiliations

1. Florida State Univ., Tallahassee, FL (United States)
2. Florida State Univ., Tallahassee, FL (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
3. Florida State Univ., Tallahassee, FL (United States). College of Medicine, Translational Science Laboratory
4. Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)

Publication Date:

Tue Sep 12 00:00:00 EDT 2023

Research Org.:

Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)

Sponsoring Org.:

USDOE Office of Science (SC), Basic Energy Sciences (BES); USDOE Office of Science (SC), Biological and Environmental Research (BER); National Science Foundation (NSF)

OSTI Identifier:

2001374

Grant/Contract Number:  

AC05-00OR22725; MCB1856502; CHE1904612

Resource Type:

Accepted Manuscript

Journal Name:

Journal of Structural Biology

Additional Journal Information:

Journal Volume: 215; Journal Issue: 4; Journal ID: ISSN 1047-8477

Publisher:

Elsevier

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; sulfur metabolism; NADPH-dependent sulfite reductase; Diflavin reductase; redox enzyme; small-angle neutron scattering; mass spectrometry