Fig. 3

Biosynthetic pathway of azoxy bond. a HPLC analysis of in vitro non-enzymatic reactions at 370 nm. Reactions were performed in 20 mM HEPES buffer (pH 7). (i) Standard 8; (ii) standard 7; (iii) standard 9; (iv) 8 in buffer for 1 min; (v) 8 with 10 eq NADH in buffer for 1 min; (vi) 8 with 10 eq NAD⁺ in buffer for 1 min; (vii) 8 with 10 eq NAD⁺ and 10 eq NADH in buffer for 1 min; (viii) 7 in buffer for 1 min; (ix) 7 with 10 eq NAD⁺ in buffer for 1 min; (x) 7 with 10 eq NADH in buffer for 1 min; (xi) 7 with 10 eq NAD⁺ and 10 eq NADH in buffer for 1 min; (xii) 7 + 8 in buffer for 1 min; (xiii) 7 + 8 with 10 eq NAD⁺ and 10 eq NADH in buffer for 1 min. b Biosynthetic scheme of 9 from 6. AzoC enzymatically oxidizes 6 to 8, and then NAD⁺/NADH induces non-enzymatic reciprocal transformation between 7 and 8. 9 is then formed during the redox reaction process. c Proposed radical biosynthetic mechanism of azoxy bond from nitroso group