Light‐Activated Electron Transfer and Catalytic Mechanism of Carnitine Oxidation by Rieske‐Type Oxygenase from Human Microbiota

Abstract Oxidation of quaternary ammonium substrate, carnitine by non‐heme iron containing Acinetobacter baumannii (Ab) oxygenase CntA/reductase CntB is implicated in the onset of human cardiovascular disease. Herein, we develop a blue‐light (365 nm) activation of NADH coupled to electron paramagnetic resonance (EPR) measurements to study electron transfer from the excited state of NADH to the oxidized, Rieske‐type, [2Fe‐2S]2+ cluster in the AbCntA oxygenase domain with and without the substrate, carnitine. Further electron transfer from one‐electron reduced, Rieske‐type [2Fe‐2S]1+ center in AbCntA‐WT to the mono‐nuclear, non‐heme iron center through the bridging glutamate E205 and subsequent catalysis occurs only in the presence of carnitine. The electron transfer process in the AbCntA‐E205A mutant is severely affected, which likely accounts for the significant loss of catalytic activity in the AbCntA‐E205A mutant. The NADH photo‐activation coupled with EPR is broadly applicable to trap reactive intermediates at low temperature and creates a new method to characterize elusive intermediates in multiple redox‐centre containing proteins.

S3 Figure S1. cw-EPR spectra of the 'AbCntA+carnitine' in the presence (top) and absence (bottom) of NADH, following photoactivation using blue-light, 365 nm for various time scale. The spectra were measured as a frozen solution at 20 K. Conditions -as described in the experimental section. Figure S2. Comparisons of cw-EPR spectra of the 'AbCntA' in the presence (red and blue traces) and absence (black trace) of substrate, carnitine, following photoactivation using blue-light, 365 nm for 30 min. The EPR spectra are expanded between 3250-3400 G (bottom) to show the overlapping signals arising from two different EPR active species and the EPR line broadening. Conditions -as described in the experimental section. Figure S3. Experimental (black trace) and simulated (red dotted line) EPR spectra of the photoactivated 'AbCntA+carnitine+NADH' measured as a frozen solution at 20 K. The spectrum was successfully simulated by considering two contributing, S = ½ spin species with the following spin-Hamiltonian parameters (also provided in Table S1 Figure S4. Experimental (black trace) and simulated (red dotted line) EPR spectra of the photoactivated 'AbCntA+NADH' measured as a frozen solution at 20 K. The spectrum was successfully with the following spin-Hamiltonian parameters (also provided in Table S1    WT+dithionite" rules out that the EPR signal at g = 2 signal was not due to dithionite. S13 Figure S12. narrow-sweep, cw-EPR spectra of dithionite reduced (red traces) and NADHphotoactivated (black traces), "AbCntA" in the presence (top) and absence (bottom) of carnitine show the formation of, one-electron reduced, Rieske, [2Fe-2S] +1 EPR signals in both samples. The two red asterisk marks indicate that the strong EPR signals arising from the quartz-impurity in the EPR tube (see Figure S11) are truncated in this region to show the effective reduction by NADHphotoactivation process. Conditions -as described in the experimental section.

S5
S14 Figure S13. cw-EPR spectra of the 'AbCntA-E205A+NADH', following photoactivation using bluelight, 365 nm for various time scale. The spectra were measured as a frozen solution at 20 K.
Conditions -as described in the experimental section. S15 Figure S14. (top) EPR spectral changes observed when "AbCntA-E205A+NADH" was annealed at the specified temperatures/times, following photoexcitation using 365 nm, blue-light for 120 minutes.
(bottom) All the spectra were normalised relative to the EPR intensity observed at 240 K (3340 G) after photoactivation of NADH for 120 minutes. The comparison of relative EPR signal of "AbCntA-E205A+NADH" observed at 275 K (~ 30 %) to that of "AbCntA-WT +NADH" at 270 K ( Figure S7; ~ 80 %), suggest that the bridging glutamate plays a critical role in stabilising the one-electron reduced, Rieske centre in the absence of carnitine. Conditions -as described in the experimental section.
S16 Figure S15. Comparisons of narrow-sweep, cw-EPR spectra of photoactivated, "AbCntA-WT+NADH" (black traces; 30 min) and "AbCntA-E205A+NADH" (red traces; 120 min) before (bottom) and after (top) annealing at 270 K (AbCntA-WT+NADH -20 min)/275 K (AbCntA-E205A+NADH; 5 min). All EPR spectra were measured as a frozen solution at 20 K. The observation of significantly reduced catalytic activity for the "AbCntA-E205A" mutant is likely due to the disruption in the electron transfer process and its incompetence to stabilise the one-electron reduced centre as shown in the comparison. Conditions -as described in the experimental section.

AUTHORS CONTRIBUTION
MS designed and conceived experiments, performed all EPR measurements and simulations, collated, analyzed and interpreted data, wrote the manuscript. MQ prepared all samples, and managed all experimental aspects of the project. YC, AC and TB secured funding and directed the overall project. All authors commented on the final manuscript.