Non-enzymatic hydrogen sulfide production from cysteine in blood is catalyzed by iron and vitamin B6

Abstract

Hydrogen sulfide (H₂S) plays important roles in metabolism and health. Its enzymatic generation from sulfur-containing amino acids (SAAs) is well characterized. However, the existence of non-enzymatic H₂S production from SAAs, the chemical mechanism, and its biological implications remain unclear. Here we present non-enzymatic H₂S production in vitro and in blood via a reaction specific for the SAA cysteine serving as substrate and requires coordinated catalysis by Vitamin B₆, pyridoxal(phosphate), and iron under physiological conditions. An initial cysteine-aldimine is formed by nucleophilic attack of the cysteine amino group to the pyridoxal(phosphate) aldehyde group. Free or heme-bound iron drives the formation of a cysteine-quinonoid, thiol group elimination, and hydrolysis of the desulfurated aldimine back to pyridoxal(phosphate). The reaction ultimately produces pyruvate, NH₃, and H₂S. This work highlights enzymatic production is inducible and robust in select tissues, whereas iron-catalyzed production contributes underappreciated basal H₂S systemically with pathophysiological implications in hemolytic, iron overload, and hemorrhagic disorders.

Keywords: Biochemistry; Haematological diseases; Physiology.

Fig. 1

Enzymatic and non-enzymatic H₂S production is tissue specific. a, b Generalized models of Vitamin B₆ (VitB₆) (PLP)-dependent enzymatic H₂S production. H₂S is generated from cysteine or homocysteine via the transsulfuration pathway enzymes, cystathionine β-synthase (CBS), and cystathionine γ-lyase (CGL) (a), or through the stepwise deamination of cysteine to 3-mercaptopyruvate (3-MP) by cysteine/asparatate aminotransferase (CAT) and release of H₂S via 3-mercaptopyruvate sulfurtransferase (3-MST) (b). c, d H₂S production from tissue extracts (c) (n = 3/group) or from plasma and red blood cells (RBCs) (d) (n = 5/group) collected from CGL WT and CGL KO mice in the presence of l-cysteine and PLP. Asterisk indicates the significance of the difference versus CGL WT; *P < 0.05. e, f H₂S production capacity of tissue extracts, plasma, and RBCs from CGL WT mice in the presence of l-cysteine and PLP ± Proteinase K (Prot. K) pretreatment as measured after 3 h incubation (e) or 16 h incubation (f), n = 3/group. Asterisk indicates the significance of the difference versus sample without Prot. K pretreatment; *P < 0.05. g Non-enzymatic H₂S production in DMEM media or DMEM/F12 media in the presence of cysteine and/or PLP; n = 3/group. Asterisk indicates the significance of the difference vs. media-only group; *P < 0.05. All data were presented as mean ± SEM

Fig. 2

Fe³⁺ and PLP coordinate to catalyze H₂S production from l-cysteine under physiological conditions. a H₂S production catalyzed by inorganic metal ions as indicated, in the reaction mixture of l-cysteine and PLP; n = 6/group. Asterisk indicates the significance of the difference versus the l-cysteine and PLP alone reaction mixture control group; *P < 0.05. b–e The effect of pH (b; n = 6/group), temperature (c; n = 5/group), O₂ (d; n = 5/group), and Fe³⁺ concentration (e; n = 6/group) on H₂S production from reaction mixtures containing l-cysteine, PLP, and Fe³⁺ (b, c, e) or the l-cysteine and PLP mixture group (d); *P < 0.05. f H₂S production in PBS ± l-cysteine ± PLP, ±Fe³⁺, and with the pretreatment of EDTA; n = 6/group. Asterisk indicates the significance of the difference vs. the l-cysteine and PLP mixture group, and pound indicates the significance of the difference between PBS ± l-cysteine ± PLP, ±Fe³⁺ with and without EDTA; *,^#P < 0.05. All data were presented as mean ± SEM

Fig. 3

Detection of headspace and dissolved H₂S catalyzed by Fe³⁺ and PLP with l-cysteine as substrate. a, b H₂S levels (parts per billion; p.p.b.) in 6 mL headspace vials detected using the Jerome J605 after (a) 1 h incubation of reaction mixture containing supraphysiological concentration of l-cysteine (10 mM), PLP (1 mM), and Fe³⁺ (50 µM); n = 3/group, or after (b) 6 h incubation of reaction mixture containing more relevant physiological concentrations of l-cysteine (500 µM), PLP (500 nM), and Fe³⁺ (50 µM); n = 3/group. Asterisk indicates the significance of the difference versus the PBS background control; *P < 0.05. c Lead acetate/lead sulfide H₂S production analysis under similar physiological conditions as in b with overnight exposure at 37 °C; n = 6/group. d, e Headspace H₂S detected in the selected ion flow tube mass spectrometry (SIFT-MS) with d quantitative targeted scan for H₂S; n = 1 for air control and NaHS groups, and n = 2 for PBS/PLP/L-Cys and PBS/PLP/L-Cys/Fe(III) groups, and e truncated mass spectrum over the range of mass-to-charge (m/z) shown in H₃O⁺ reagent ion, measuring the product ions generated in the reaction with H₃O⁺, being the m/z 35 product ion. The precursor ion signals (H₃O⁺·(H₂O)_0,1,2 as appropriate) and the product ion signals are indicated. The concentrations of the trace gases are given in parts per million (p.p.m.). For full mass spectrum over the m/z, please see Supplementary Fig. 3c, d. f Time-dependent detection of dissolved H₂S using the fluorogenic AzMC probe (Exc 350 nm/Emi 445 nm) from various reaction mixtures; n = 6/group. All data were presented as mean ± SEM

Fig. 4

l- and d-Cysteine, but not other SAAs, act as substrate for rapid H₂S production catalyzed by Fe³⁺ and PLP. a Chemical structure of the sulfur amino acids (SAAs). b H₂S production capacity as determined by the lead acetate/lead sulfide method after 2.5 h incubation with different SAAs as substrate catalyzed by Fe³⁺ and PLP; n = 6/group. Asterisk indicates the significance of the difference versus the l-cysteine and PLP-alone control mixture group; *P < 0.05. c Chemical structure of N-acetylcysteine (NAC). d H₂S production capacity with NAC as substrate in the presence of Fe³⁺ and PLP; n = 4/group. e Chemical structures of PLP and pyridoxal. f, g Selectivity of PLP and pyridoxal as co-factors for enzymatic H₂S production in liver extract (f); n = 6/group, and non-enzymatic iron-catalyzed H₂S production (g); n = 6/group. Asterisk indicates the significance of the difference versus the control PLP/Pyridoxal-null reaction mixture 0; *P < 0.05. Data were presented as mean ± SEM

Fig. 5

Mechanistic model for Fe³⁺- and PLP-catalyzed H₂S production. a Proposed reaction model for H₂S production from cysteine, PLP, and Fe³⁺: (i) the nucleophilic attack by the free amino group of cysteine on PLP forms a Schiff base, the cysteine-aldimine; (ii) deprotonation at α-position of cysteine leads to the formation of a quinonoid intermediate; (iii) the elimination of ‒SH group catalyzed by Fe³⁺; and (iv) the desulfurated aldimine is hydrolyzed to produce pyruvate, NH₃, and regenerate PLP; (v) in the absence of Fe³⁺, a thiazolidine ring is formed from cysteine-aldimine product from step (i), offering UV/VIS peak absorbance at 333 nm. b AOAA, but not PAG, inhibits Fe³⁺- and PLP-catalyzed H₂S production; n = 3/group. Asterisk indicates the significance of the difference vs. the l-cysteine, Fe³⁺, and PLP control reaction mixture group; *P < 0.05. c Detection of PLP Schiff base by UV/VIS spectrophotometer. Absorbance of PLP in the absence or presence of tested sulfur amino acids was measured in the spectrum of 310–435 nm. The formation of Schiff base at ~330 nm is indicated by the shift of the peak with the addition of tested sulfur amino acids from ~390 nm. d Changes in absorbance in reaction mixture of PLP ± cysteine ± Fe³⁺ with reaction time. Loss of absorbance at 330 nm and gain at 390 nm after 24 h incubation in reaction mixture of cysteine with Fe³⁺ and PLP indicate Fe³⁺ consumes the substrate of cysteine and PLP is regenerated. e Pyruvate formation in reaction mixture of cysteine and PLP, ±FeCl₃; n = 4/group. Data are measured colorimetrically at absorbance of 570 nm showing quantity (nmol) per ml H₂S reaction mixture. All data were presented as mean ± SEM

Fig. 6

Heme-bound iron catalyzes H₂S production in vitro and in blood/plasma ex vivo. a, b Hemin (a; n = 6/group) and ferritin (b; n = 5/group) dose dependently catalyze H₂S production from l-cysteine and PLP. Asterisk indicates the significance of the difference versus the l-cysteine and PLP control group; *P < 0.05. c Iron-dependent H₂S production from RBC lysate ± l-cysteine and PLP reaction mix, ±DTPA; n = 5/group. Asterisk indicates the significance of the difference between indicated groups; *P < 0.05. d, e H₂S production from RBC lysate (d; n = 4/group) or plasma (e; n = 3/group) ± theoretical inhibitors PAG and AOAA and ±iron chelator DTPA with the addition of l-cysteine and PLP. Asterisk indicates the significance of the difference versus the l-cysteine and PLP control group; *P < 0.05. f H₂S production from the reaction mixture of whole or lysed RBCs, l-cysteine, and PLP ± pretreatment of DTPA and ±Prot. K); n = 3/group. Asterisk indicates the significance of the difference versus non-lysed RBCs with no DTPA or Prot. K pretreatment; *P < 0.05. All data were presented as mean ± SEM

Fig. 7

Experimental model of red blood cell state and tissue integrity impacting iron-catalyzed non-enzymatic H₂S production. Iron in red blood cells and tissues catalyzes the production of H₂S in coordination with VitB₆ from the sulfur-containing amino acid cysteine at physiological temperatures, pH, and oxygen conditions. Multiple biologically relevant forms of VitB_6, PLP or pyridoxine, and iron, free or bound Fe³⁺/Fe²⁺, served as catalysts. Upon hemolysis, tissue damage, and/or degradation of iron- and heme-containing proteins, the catalytic potential of iron is increased and more H₂S is produced. The biological significances of this increased H₂S production, particularly in the context of hemolytic anemias and crises, are yet to be determined