Interaction of heme and heme–hemopexin with an extracellular oxidant system used to measure cell growth-associated plasma membrane electron transport

Abstract

Since redox active metals are often transported across membranes into cells in the reduced state, we have investigated whether exogenous ferri-heme or heme bound to hemopexin (HPX), which delivers heme to cells via receptor-mediated endocytosis, interact with a cell growth-associated plasma membrane electron transport (PMET) pathway. PMET reduces the cell-impermeable tetrazolium salt, WST-1, in the presence of the mandatory low potential intermediate electron acceptor, mPMS. In human promyelocytic (HL60) cells, protoheme (iron protoporphyrin IX; 2,4-vinyl), mesoheme (2,4-ethyl) and deuteroheme (2,4-H) inhibited reduction of WST-1/mPMS in a saturable manner supporting interaction with a finite number of high affinity acceptor sites (Kd 221 nM for naturally occurring protoheme). A requirement for the redox-active iron was shown using gallium-protoporphyrin IX (PPIX) and tin-PPIX. Heme-hemopexin, but not apo-hemopexin, also inhibited WST-1 reduction, and copper was required. Importantly, since neither heme nor heme-hemopexin replace mPMS as an intermediate electron acceptor and since inhibition of WST-1/mPMS reduction requires living cells, the experimental evidence supports the view that heme and heme-hemopexin interact with electrons from PMET. We therefore propose that heme and heme-hemopexin are natural substrates for this growth-associated electron transfer across the plasma membrane.

Hemin causes mitochondrial dysfunction in endothelial cells through promoting lipid peroxidation: the protective role of autophagy

“…DMSO was used as a vehicle control, such that the same volume of vehicle was added to all treatment groups. Hemin chloride was dissolved in DMSO and verified to be in the monomeric form by measuring the absorbance at 406 nm (⑀ ϭ 170 mM/cm) (73). Hemin was added directly from the DMSO stock to the medium before addition to cells.…”

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“…In a paper by Rish et al [32] it was demonstrated that highly proliferative cells are characterized by a plasma membrane electron transport (PMET) that enables cells to transfer electrons from intracellular reductants, like NADH, to extracellular electron acceptors. Among electron acceptors, Rish et al indicated the heme-Hx complex as one of the best physiological candidates for this function.…”

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“…93 PMOR is measured using two substrates: reduction of the water soluble tetrazolium salt, WST-1 [2,(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium), in the presence of an intermediate electron acceptor [IEA; mPMS (1-methoxy-5-methylphenazinium methylsulfate)] 92 The redox potential of heme-HPX is ~50mV, and evidence that heme-HPX is a substrate for PMOR has been obtained in human promyelocytic HL60 cells and linked to the LRP1-independent low-affinity, high-capacity uptake system of heme-HPX. 3,19 Additional evidence links PMOR/PMET and redox cycling of copper with heme-HPX endocytosis. 19 If there is incomplete reduction of oxygen, then an oxidase component may generate superoxide from which H 2 O 2 is derived.…”

“…3,19 Additional evidence links PMOR/PMET and redox cycling of copper with heme-HPX endocytosis. 19 If there is incomplete reduction of oxygen, then an oxidase component may generate superoxide from which H 2 O 2 is derived. In the presence of reduced copper or iron, the hydroxyl radical and carbonyls will be produced.…”

“…The sole possible site for which there is experimental evidence for heme reduction is, like that for heme-HPX described above, at the plasma membrane via electron transport by PMET/PMOR, for which heme is a substrate in vitro. 19 Alternatively, damage comes from catalytic iron that may be released when the heme ring is destroyed in the presence of H 2 O 2 . If this iron is reduced presumably by superoxide, then the highly reactive hydroxyl radical is generated from H 2 O 2 by Fenton chemistry.…”