Enzymes accelerate biological reactions by up to millions of times; oxidation enzymes are a subgroup that transfer electrons in redox reactions. Theorell crystallized oxidation enzymes from liver and blood and used absorption spectroscopy and chemical probes to identify their active sites and coenzymes. He performed the first quantitative analysis of how alcohol dehydrogenase binds the coenzyme NAD⁺, revealing that the enzymatic reaction proceeds in discrete steps. These insights explained how respiration, fermentation and drug detoxification share common mechanistic principles. His results showed that defects in oxidation enzymes can produce vitamin deficiencies, anemia or alcohol-metabolism disorders, opening paths for diagnostic tests and therapies. Current standards for measuring enzyme activity in biochemistry and medicine trace back directly to Theorell’s methods.

Between the 1930s and 1950s Theorell purified heme enzymes such as catalase and peroxidase as well as flavoproteins, and followed their reaction intermediates by low-temperature absorption spectroscopy combined with pulse addition of oxidants. He identified the high-valent iron-oxo species generated during the catalase catalytic cycle—now known as Compound I—and formulated the mechanism by which oxygen radicals are removed. For alcohol dehydrogenase he demonstrated that the pyridine nucleotide NAD⁺ binds to the enzyme in a defined order, forms a hydride-transfer intermediate, and employed initial-rate kinetics to discriminate between sequential and ping-pong mechanisms. By integrating data on redox potentials, kinetic isotope effects and pH dependence, he pioneered the quantitative description of enzymatic mechanisms. He further proved the stereoselectivity of substrate binding and built stereochemical models of the active site, laying groundwork for modern structural biology and enzyme engineering. The understanding of oxidation enzymes was later extended to the mitochondrial electron-transport chain and drug-metabolizing enzymes, exerting profound influence on both bioenergetics and pathobiochemistry.

By combining differential absorption spectroscopy with stopped-flow measurements, Theorell monitored the Fe(III)→Fe(IV)=O transition of the catalase active site and the associated porphyrin π→π* shifts with millisecond resolution. The resulting concept of Compound I became a unifying paradigm for heme enzymes such as peroxidases and cytochrome P450. In alcohol dehydrogenase he quantitatively tracked the 320-nm NAD⁺ bleaching and Zn²⁺ active-site changes, proving that hydride transfer proceeds via a concerted two-electron pathway. His Michaelis–Menten analyses were pioneering in experimentally discriminating the ordered-complex from the ping-pong Bi-Bi model. For flavin enzymes he used pKa shifts and extinction changes to identify the flavin–hydroxyanion transition state and rationalized the stoichiometry of the Warburg–Theorell oxidation. The resulting data agreed with ESR and low-temperature crystallography, establishing that charge redistribution and stereochemistry at redox centers govern the rate-limiting step. He further proposed an induced-fit model that couples conformational changes with chemistry. These insights are still cited in discussions of PCET and single-particle cryo-EM, extending far beyond classical enzymology.