The RNA-guided Cas9 endonuclease from Staphylococcus aureus (SauCas9) can catalyze multiple-turnover reactions whereas Cas9 from Streptococcus pyogenes (SpyCas9) is a single-turnover enzyme. Here we dissect the mechanism of multiple-turnover catalysis by SauCas9 and elucidate its molecular basis. We show that the multiple-turnover catalysis does not require more than stoichiometric RNA guides to Cas9 nuclease. Rather, the RNA-guide loaded ribonucleoprotein (RNP) is the reactive unity that is slowly released from product and recycled in the subsequent reaction. The mechanism that RNP is recycled for multiple-turnover reaction entails the unwinding of the RNA:DNA duplex in the R-loop. We argue that DNA rehybridization is required for RNP release by supplementing the energy cost in the process. Indeed, turnover is arrested when DNA rehybridization is suppressed. Further, under higher salt conditions, both SauCas9 and SpyCas9 showed increased turnover, and engineered SpyCas9 nucleases that form fewer direct or hydrogen bonding interactions with target DNA became multiple-turnover enzymes. Thus, these results indicate that for both SpyCas9 and SauCas9, turnover is determined by the energetic balance of the post-chemistry RNP-DNA interaction. Due to the conserved protein core folds, the mechanism underpinning turnover we establish here is likely operant in all Cas9 nucleases.

In mammals, iron(II)‐ and 2‐oxoglutarate‐dependent (Fe/2OG) dioxygenases have roles in oxygen and body‐mass homeostasis, connective tissue synthesis, and control of transcription and epigenetic inheritance.1 Related enzymes in plants, fungi, and bacteria enable diverse biosynthetic pathways to valuable natural‐product drugs by catalyzing halogenation, epimerization, desaturation, cyclization, ring‐opening/expansion, and endoperoxidation reactions; some of these enzymes even catalyze multiple reaction types within the same pathway!2 Almost every enzyme in this class initiates its reaction by using a common oxoiron(IV) (ferryl) intermediate, first demonstrated by the Penn State group in 2003,3,4 to abstract a hydrogen atom from its target substrate (Figure 1). The iron(II)‐chelating co‐substrate, 2OG, is oxidatively decarboxylated to a succinate ligand in formation of the ferryl complex. To mediate an outcome other than hydroxylation, an enzyme must avoid what can be a facile “oxygen rebound,” Groves term for the coupling of the substrate radical with the iron‐coordinated oxygen just after it abstracts the hydrogen. By direct biophysical observation and “metallomimicry” of intermediates, we have rationalized several of these outcomes in terms of the alternative fates of the substrate radical and the structural features of the enzyme that avert oxygen rebound to enable these alternative fates.5‐11 Very recently, we explained how the most unusual Fe/2OG oxygenase discovered to date, the microbial ethylene‐forming enzyme (EFE), branches from the canonical pathway even before the ferryl intermediate is formed (Figure 2),12,13 leading to global fragmentation of 2OG to three carbon dioxide equivalents and ethylene, a reaction that requires but does not transform the amino acid L‐arginine. The elucidation of the unusual “radical‐polar‐crossover” mechanism of EFE13 significantly expands the paradigm of 2OG‐assisted dioxygen activation pathways and suggests unexpected uses of EFE in biotechnology. 1. Hausinger, R. P. Crit. Rev. Biochem. Mol. Biol., 2004, 39, 21–68. 2. Bollinger, J. M., Jr., et al. 2015 in RSC Metallobiology Series No. 3. R.P. Hausinger and C.J. Schofield, eds. pp. 95‐122. Royal Society of Chemistry, Washington, D.C. 3. Price, J. C., et al. Biochemistry,2003,42,7497‐7508. 4. Price, J. C., et al. J. Am. Chem. Soc., 2003, 125, 13008‐13009. 5. Matthews, M. L., et al. Proc. Natl. Acad. Sci. USA, 2009, 106, 17723‐17728. 6. Chang, W.‐c., et al. Science 2014, 343, 1140‐1143. 7. Martinie, R. J., et. al. J. Am. Chem. Soc. 2015,137, 6912‐6919. 8. Martinie, R. J., et al. Inorg. Chem. 2017, 56, 13382‐13389. 9. Dunham, N. P. et al. J. Am. Chem. Soc. 2018, 140, 7116‐7128. 10. Dunham N. P., et al. J. Am. Chem. Soc. 2019, 141, 9964‐9979. 11. Pan, J., et al. J. Am. Chem. Soc.2019, 141, 15153−15165. 12. Copeland, R. A., et al. J. Am. Chem. Soc. 2021, 143, 2293‐2303. 13. Copeland, R. A., et al. Science2021, 373, 1489‐1493.