Pyruvate dehydrogenase operates as an intramolecular nitroxyl generator during macrophage metabolic reprogramming

M1 macrophages enter a glycolytic state when endogenous nitric oxide (NO) reprograms mitochondrial metabolism by limiting aconitase 2 and pyruvate dehydrogenase (PDH) activity. Here, we provide evidence that NO targets the PDH complex by using lipoate to generate nitroxyl (HNO). PDH E2-associated lipoate is modified in NO-rich macrophages while the PDH E3 enzyme, also known as dihydrolipoamide dehydrogenase (DLD), is irreversibly inhibited. Mechanistically, we show that lipoate facilitates NO-mediated production of HNO, which interacts with thiols forming irreversible modifications including sulfinamide. In addition, we reveal a macrophage signature of proteins with reduction-resistant modifications, including in DLD, and identify potential HNO targets. Consistently, DLD enzyme is modified in an HNO-dependent manner at Cys477 and Cys484, and molecular modeling and mutagenesis show these modifications impair the formation of DLD homodimers. In conclusion, our work demonstrates that HNO is produced physiologically. Moreover, the production of HNO is dependent on the lipoate-rich PDH complex facilitating irreversible modifications that are critical to NO-dependent metabolic rewiring.

-Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy

Human research participants
Policy information about studies involving human research participants and Sex and Gender in Research.
Reporting on sex and gender Population characteristics

Recruitment
Ethics oversight Note that full information on the approval of the study protocol must also be provided in the manuscript.

Field-specific reporting
Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

Life sciences
Behavioural & social sciences Ecological, evolutionary & environmental sciences For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative. Reporting for specific materials, systems and methods All data supporting the findings of this study are available with the article, and can also be obtained from the corresponding author. Publicly available crystallized structure of the human DLD (PDB_ID: 3rnm) and hDLD crystallized in complex with FAD and NAD+ (1zmd.pdb) were used in this study. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the MASSive partner repository under accession code MSV000092336. Source data are provided with this paper.
3-9 mice per group were used for animal studies. No prior sample size calculation was performed. Group sizes for animal studied were established considering previous experience with yields of cell numbers and biological matrices. No statistical methods were used to predetermine sample size. Sample sizes were determined based on prior studies with similar experimental design and on the known variability of the assay, balancing statistic robustness with resource availability. The number of mice that were used for each experiment was determined to give reliable and robust conclusions. The number of mice per group for each experiment are indicated in figure legends, and depicted as individual dots in graphs. Animal experiments were repeated at least three times.
No data exclusions were made.
Results of experiments are based on representative results of at least 3 independent bone marrow derived macrophage cultures (for in vitro experiments)/cohorts of mice (for in vivo experiments) following the same protocol. All findings were replicated successfully.
Animals were randomized by age for bone marrow extractions. For in vivo studies, mice were also randomized by bodyweight and sex prior to challenge with LPS. For in vitro experiments samples were randomly allocated into experimental groups.
Investigators were blinded as to the experimental groups mice were allocated. Only animal technical staff who were performing the injections were aware of the allocation of mice to each group. Investigators performing analysis of samples for mass spectrometry (proteomics and metabolomics) were blinded to the allocation of mice. Investigators were not blinded for the in vitro experiments because the treatment groups were labeled, however all experiments were objective and conclusions based on multiple technical replicates and statistical significance.