Healthy guts exclude oxygen: Normally, the lumen of the colon lacks oxygen. Fastidiously anaerobic butyrate-producing bacteria thrive in the colon; by ablating these organisms, antibiotic treatment removes butyrate. Byndloss et al. discovered that loss of butyrate deranges metabolic signaling in gut cells (see the Perspective by Cani). This induces nitric oxidase to generate nitrate in the lumen and disables β-oxidation in epithelial cells that would otherwise mop up stray oxygen before it enters the colon. Simultaneously, regulatory T cells retreat, and inflammation is unchecked, which contributes yet more oxygen species to the colon. Then, facultative aerobic pathogens, such as Escherichia coli and Salmonella enterica , can take advantage of the altered environment and outgrow any antibiotic-crippled and benign anaerobes. Science , this issue p. 570 ; see also p. 548

The production of NO^⋅ by mitochondria was investigated by electron paramagnetic resonance using the spin-trapping technique, and by the oxidation of oxymyoglobin. Percoll-purified rat liver mitochondria exhibited a negligible contamination with other subcellular fractions (1–4%) and high degree of functionality (respiratory control ratio = 5–6). Toluene-permeabilized mitochondria, mitochondrial homogenates, and a crude preparation of nitric oxide synthase (NOS) incubated with the spin trapN-methyl-d-glucamine-dithiocarbamate-Fe^IIproduced a signal ascribed to the NO^⋅ spin adduct (g = 2.04; a _N = 12.5 G). The intensity of the signal increased with time, protein concentration, andl-Arg, and decreased with the addition of the NOS inhibitorN ^G-monomethyl-l-arginine. Intact mitochondria, mitochondrial homogenates, and submitochondrial particles produced NO^⋅ (followed by the oxidation of oxymyoglobin) at rates of 1.4, 4.9, and 7.1 nmol NO^⋅ × (min·mg protein)⁻¹, respectively, with aK _m for l-Arg of 5–7 μm. Comparison of the rates of NO^⋅ production obtained with homogenates and submitochondrial particles indicated that most of the enzymatic activity was localized in the mitochondrial inner membrane. This study demonstrates that mitochondria are a source of NO^⋅, the production of which may effect energy metabolism, O₂consumption, and O₂ free radical formation.

Autism spectrum disorders (ASD) are characterized by impaired social interaction, problems with verbal and nonverbal communication, and repetitive or severely restricted activities and interests.¹ Limited scientific advances have been made regarding the causes of autism, with general agreement that both genetic and environmental factors contribute to this disorder.

We reported that the generation of nitric oxide by mitochondria is catalyzed by a constitutive, mitochondrial nitric-oxide synthase (mtNOS). Given that this production may establish the basis for a novel regulatory pathway of energy metabolism, oxygen consumption, and oxygen free radical production, it becomes imperative to identify unequivocally and characterize this enzyme to provide a basis for its regulation. The mitochondrial localization of mtNOS was supported by following the hepatic distribution of mtNOS, immunoblotting submitochondrial fractions, and immunohistochemistry of liver tissues. mtNOS was identified as brain NOS alpha by various methods (mass spectrometry of proteolytic fragments, amino acid analysis, molecular weight, pI, and analysis of PCR fragments), excluding the occurrence of a novel isoform or other splice variants. Distribution of mtNOS transcript indicated its occurrence in liver, brain, heart, muscle, kidney, lung, testis, and spleen. In contrast to brain NOS, mtNOS has two post-translational modifications: acylation with myristic acid and phosphorylation at the C terminus. The former modification is a reversible and post-translational process, which may serve for subcellular targeting or membrane anchoring. The latter modification could be linked to enzymatic regulation. These results are discussed in terms of the role that nitric oxide may have in cellular bioenergetics.

The biosynthesis of nitric oxide (NO^⋅) in different cell types occurs concomitantly with the conversion ofl-arginine to l-citrulline by the enzyme nitric-oxide synthase (NOS). NO^⋅ has been identified as a major participant in a number of basic physiological functions such as neurotransmission, vasodilation, and immune response. At the subcellular level, mitochondria have been identified as targets for NO^⋅; however, to date, no unambiguous evidence has been presented to identify these organelles as sources of NO^⋅. In this study, a NOS was isolated to homogeneity from Percoll-purified rat liver mitochondria. Kinetic parameters, molecular weight, requirement of cofactors, and cross-reactivity to monoclonal antibodies against macrophage NOS suggest similarities to the inducible form. However, the constitutive expression of the mitochondrial enzyme and its main membrane localization indicate the presence of either a distinctive isoform or a macrophage isoform containing posttranslational modifications that lead to different subcellular compartments. The detection of NADPH-oxidizing activities and a production of superoxide anion catalyzed by mtNOS and recombinant cytochrome P₄₅₀reductase were consistent with the sequence homology reported for these two proteins. Given the role of NO^⋅ as cellular transmitter, messenger, or regulator, the presence of a functionally active mitochondrial NOS may have important implications for the intermediary metabolism.