Lactoperoxidase catalytically oxidize hydrogen sulfide via intermediate formation of sulfheme derivatives.

The biological chemistry of hydrogen sulfide (H2S) with physiologically important heme proteins is in the focus of redox biology research. In this study, we investigated the interactions of lactoperoxidase (LPO) with H2S in the presence and absence of molecular dioxygen (O2) or hydrogen peroxide (H2O2). Under anaerobic conditions, native LPO forms no heme-H2S complex upon sulfide exposure. However, under aerobic conditions or in the presence of H2O2 the formation of both ferrous and ferric sulfheme (sulfLPO) derivatives was observed based on the appearances of their characteristic optical absorptions at 638 nm and 727 nm, respectively. Interestingly, we demonstrate that LPO can catalytically oxidize H2S by H2O2 via intermediate formation of relatively short-lived ferrous and ferric sulfLPO derivatives. Pilot product analyses suggested that the turnover process generates oxidized sulfide species, which include sulfate S O 4 2 - and inorganic polysulfides ( H S x - ; x = 2-5). These results indicated that H2S can serve as a non-classical LPO substrate by inducing a reversible sulfheme-like modification of the heme porphyrin ring during turnover. Furthermore, electron paramagnetic resonance data suggest that H2S can act as a scavenger of H2O2 in the presence of LPO without detectable formation of any carbon-centered protein radical species, suggesting that H2S might be capable of protecting the enzyme from radical-mediated damage. We propose possible mechanisms, which explain our results as well as contrasting observations with other heme proteins, where either no sulfheme formation was observed or the generation of sulfheme derivatives provided a dead end for enzyme functions.

Interactions of reactive sulfur species with metalloproteins.

Reactive sulfur species (RSS) entail a diverse family of sulfur derivatives that have emerged as important effector molecules in H2S-mediated biological events. RSS (including H2S) can exert their biological roles via widespread interactions with metalloproteins. Metalloproteins are essential components along the metabolic route of oxygen in the body, from the transport and storage of O2, through cellular respiration, to the maintenance of redox homeostasis by elimination of reactive oxygen species (ROS). Moreover, heme peroxidases contribute to immune defense by killing pathogens using oxygen-derived H2O2 as a precursor for stronger oxidants. Coordination and redox reactions with metal centers are primary means of RSS to alter fundamental cellular functions. In addition to RSS-mediated metalloprotein functions, the reduction of high-valent metal centers by RSS results in radical formation and opens the way for subsequent per- and polysulfide formation, which may have implications in cellular protection against oxidative stress and in redox signaling. Furthermore, recent findings pointed out the potential role of RSS as substrates for mitochondrial energy production and their cytoprotective capacity, with the involvement of metalloproteins. The current review summarizes the interactions of RSS with protein metal centers and their biological implications with special emphasis on mechanistic aspects, sulfide-mediated signaling, and pathophysiological consequences. A deeper understanding of the biological actions of reactive sulfur species on a molecular level is primordial in H2S-related drug development and the advancement of redox medicine.

Overview on hydrogen sulfide-mediated suppression of vascular calcification and hemoglobin/heme-mediated vascular damage in atherosclerosis.

Vulnerable atherosclerotic plaques with hemorrhage considerably contribute to cardiovascular morbidity and mortality. Calcification is the main characteristic of advanced atherosclerotic lesions and calcified aortic valve disease (CAVD). Lyses of red blood cells and hemoglobin (Hb) release occur in human hemorrhagic complicated lesions. During the interaction of cell-free Hb with plaque constituents, Hb is oxidized to ferric and ferryl states accompanied by oxidative changes of the globin moieties and heme release. Accumulation of both ferryl-Hb and metHb has been observed in atherosclerotic plaques. The oxidation hotspots in the globin chain are the cysteine and tyrosine amino acids associated with the generation of Hb dimers, tetramers and polymers. Moreover, fragmentation of Hb occurs leading to the formation of globin-derived peptides. A series of these pro-atherogenic cellular responses can be suppressed by hydrogen sulfide (H2S). Since H2S has been explored to exhibit a wide range of physiologic functions to maintain vascular homeostasis, it is not surprising that H2S may play beneficial effects in the progression of atherosclerosis. In the present review, we summarize the findings about the effects of H2S on atherosclerosis and CAVD with a special emphasis on the oxidation of Hb/heme in atherosclerotic plaque development and vascular calcification.

Measurements for Sulfide-Mediated Inhibition of Myeloperoxidase Activity.

Oxidative stress-alleviating and inflammation-mediatory functions of hydrogen sulfide were reported to be key features of its biological actions. However, the underlying molecular mechanisms of these biological observations are not fully understood. In conditions where sulfide was proposed to be protective against oxidative stress- or inflammation-induced tissue damage (e.g., reperfusion injury, atherosclerosis, vascular inflammation), the reactive oxidant-producing function of a key neutrophil enzyme, myeloperoxidase, was reported to be a protagonist on the detrimental side. We recently described favorable interactions between sulfide and myeloperoxidase and proposed that the potent inhibition of myeloperoxidase activities could contribute to sulfide's beneficial functions in a number of cardiovascular pathologies. Our chapter is dedicated to aid future studies and drug development endeavors in this area by providing methodological guidance on how to assess the inhibitory potential of sulfide on myeloperoxidase enzymatic activities in isolated protein systems, in neutrophil homogenates, and in live neutrophil preparations.

Mechanisms of myeloperoxidase catalyzed oxidation of H2S by H2O2 or O2 to produce potent protein Cys-polysulfide-inducing species.

The interaction of heme proteins with hydrogen sulfide is gaining attention as an important element in sulfide-mediated protection against oxidative stress and in regulation of redox signaling. In our previous study we reported the efficient reversible inhibition of myeloperoxidase (MPO) activity by sulfide and the kinetics of the reactions of sulfide with ferric MPO, Compound I and Compound II. Here we provide several lines of evidence that a central intermediate species in the turnover of MPO by sulfide is the Compound III state. Compound III is formed in the reactions of sulfide with ferric or ferrous MPO in the presence of oxygen or via the reductions of Compound I or Compound II by sulfide. The regeneration of active ferric MPO from Compound III is slow - representing the rate-limiting step during turnover - but facilitated by ascorbate or superoxide dismutase. These catalytic cycles produce inorganic sulfane sulfur species, which were shown to promote protein Cys persulfidation. Based on compiling experimental data we propose that in contrast to hemoglobin, myoglobin, catalase or lactoperoxidase the formation of a sulfheme derivative in the oxidative interactions of sulfide with MPO is not a major pathway. Using the Met243Val mutant we demonstrated that the sulfonium ion linkage of the Met243 sulfur to the heme pyrrole ring A, which is a unique feature of MPO, is pivotal in the catalytic oxidation of sulfide via Compound III. The proposed novel MPO-catalyzed sulfide oxidation model does not require the initial presence of hydrogen peroxide, only oxygen to provide a slow flux of sulfane sulfur species generation, which could be important in sulfide-mediated endogenous signaling. Furthermore, peroxide-induced formation of sulfane sulfur species by MPO may have a role in protection of regulatory or functional Cys residues during (for example neutrophil induced) inflammatory oxidative stress.