# Enzymology of the Reductive Acetyl-CoA Pathway

> **NIH NIH R37** · UNIVERSITY OF MICHIGAN AT ANN ARBOR · 2021 · $175,698

## Abstract

Abstract
This proposal focuses on mechanistic studies of the key enzymes in the Wood-Ljungdahl pathway of
CO/CO2 fixation and acetyl-CoA synthesis. These enzymes are CO dehydrogenase (CODH), acetyl-CoA
synthase (ACS), the corrinoid iron-sulfur protein (CFeSP) and its cognate methyltransferase (MeTr), and
pyruvate ferredoxin oxidoreductase (PFOR). Studies of this pathway will continue to enrich the areas of
microbiology, biochemistry and bioinorganic chemistry by revealing significant new insights into the
structures of macromolecular channels, previously unknown metal clusters, unusual roles of low-valent
nickel and cobalt ions as nucleophiles, novel biochemical roles of Coenzyme A, organometallic
complexes as intermediates in enzymatic catalysis, and large conformational movements that permit
active sites to interface with multiple binding partners and redox mediators. We will use spectroscopic,
mutagenesis and kinetic experiments to characterize the catalytic mechanisms of CODH and ACS, to
elucidate the properties of hydrophobic pockets in the CO channel that connect the CODH and ACS
active sites and provide insight into the conformational changes that coordinate opening and closing of
the gas channel. For ACS, we have recently developed methods to generate nearly stoichiometric amounts
of each intermediate in the catalytic cycle and plan to determine their structural and electronic properties.
In studies of the CFeSP and MeTr, we plan to elucidate the protein-protein complexes and conformational
changes that drive methyl and electron transfer. During one catalytic cycle, the C-terminal B12-binding
domain (CTD) of the CFeSP must interface with MeTr, the small subunit of the CFeSP and the A-cluster
of ACS, as well as the FeS domain of the CFeSP when reductive activation is required. We will
determine the crystal structures and perform small angle X-ray scattering experiments of the key
complexes between the CFeSP and its binding partners. We also will perform mechanistic and structural
studies of chimeric constructs in which the CTD is linked to each of its binding partners and of these
separately expressed domains. In studies of PFOR (and its CoA-independent homolog, oxalate
oxidoreductase), we plan to characterize the complex between CODH and PFOR and its role in CO2
channeling and electron transfer. We also will elucidate the novel role of CoA in causing a 100,000-fold
increase in rate of electron transfer from a substrate-induced radical intermediate to an integral iron-sulfur
cluster.

## Key facts

- **NIH application ID:** 10386087
- **Project number:** 3R37GM039451-35S1
- **Recipient organization:** UNIVERSITY OF MICHIGAN AT ANN ARBOR
- **Principal Investigator:** Stephen Wiley Ragsdale
- **Activity code:** R37 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $175,698
- **Award type:** 3
- **Project period:** 1991-08-01 → 2023-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10386087

## Citation

> US National Institutes of Health, RePORTER application 10386087, Enzymology of the Reductive Acetyl-CoA Pathway (3R37GM039451-35S1). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10386087. Licensed CC0.

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