Catalytic enhancement of the heme-based oxygen-sensing phosphodiesterase EcDOS by hydrogen sulfide is caused by changes in heme coordination structure

Abstract

EcDOS is a heme-based O2-sensing phosphodiesterase in which O2 binding to the heme iron complex in the N-terminal domain substantially enhances catalysis toward cyclic-di-GMP, which occurs in the C-terminal domain. Here, we found that hydrogen sulfide enhances the catalytic activity of full-length EcDOS, possibly owing to the admixture of 6-coordinated heme Fe(III)-SH(-) and Fe(II)-O2 complexes generated during the reaction. Alanine substitution at Met95, the axial ligand for the heme Fe(II) complex, converted the heme Fe(III) complex into the heme Fe(III)-SH(-) complex, but the addition of Na2S did not further reduce it to the heme Fe(II) complex of the Met95Ala mutant, and no subsequent formation of the heme Fe(II)-O2 complex was observed. In contrast, a Met95His mutant formed a stable heme Fe(II)-O2 complex in response to the same treatment. An Arg97Glu mutant, containing a glutamate substitution at the amino acid that interacts with O2 in the heme Fe(II)-O2 complex, formed a stable heme Fe(II) complex in response to Na2S, but this complex failed to bind O2. Interestingly, the addition of Na2S promoted formation of verdoheme (oxygen-incorporated, modified protoporphyrin IX) in an Arg97Ile mutant. Catalytic enhancement by Na2S was similar for Met95 mutants and the wild type, but significantly lower for the Arg97 mutants. Thus, this study shows the first isolation of spectrometrically separated, stable heme Fe(III)-SH(-), heme Fe(II) and heme Fe(II)-O2 complexes of full-length EcDOS with Na2S, and confirms that external-ligand-bound, 6-coordinated heme Fe(III)-SH(-) or heme Fe(II)-O2 complexes critically contribute to the Na2S-induced catalytic enhancement of EcDOS.

Gaseous O2, NO, and CO in Signal Transduction: Structure and Function Relationships of Heme-Based Gas Sensors and Heme-Redox Sensors

“…H 2 S-induced heme modification. The mutations Arg97Ala and Arg97Ile, corresponding to the direct O 2 -binding residues on the heme distal side, modify the porphyrin ring and cause formation of verdoheme, an oxygen-incorporated porphyrin, in the presence of excess H 2 S. , Sulfheme, a well-known sulfur-incorporating porphyrin, is not formed under these conditions, unlike the case for hemoglobin and myoglobin.…”

“…The Arg97Ile and Arg97Ala mutants of Ec DOS form verdoheme, an oxygen-incorporated protoporphyrin IX iron complex, after H 2 S treatment. , Mutations at Arg97, the O 2 -interacting site in the heme Fe­(II)–O 2 complex of Ec DOS, would generate considerable amounts of H 2 O 2 due to the instability of the heme Fe­(II)–O 2 complex in the presence of H 2 S. H 2 O 2 would likely form a heme Fe­(III)–OOH complex that, with the aid of the water molecule near the heme environment, would help to catalyze the verdoheme-formation reaction. − Contrary to expectations, sulfheme was not formed under these conditions.…”

“…Notably, however, catalytic activity of the heme-based oxygensensing EcDOS PDE is substantially enhanced by the addition of H 2 S under aerobic conditions as already shown in the EcDOS section (Figure 13). 158,160 Although this enhancement is mainly caused by changes in the coordination structure of the heme iron complex in the enzyme, it is unlikely that EcDOS is a heme-based H 2 S sensor, because the H 2 S concentration needed to stimulate catalysis is in a physiologically irrelevant (∼100 μM) range. However, it is assumed that deep-sea marine organisms living near volcanic hypothermal vents use H 2 Sassociated biochemistry and physiology.…”

The Novel Role of Heme in Health and Diseases – Heme-Containing Sensor Proteins

“…Během posledních osmi let jsme charakterizovali a vysvětlili vztahy mezi strukturou a funkcí a zaměřili se na přenos signálu v případě tří základních modelových prokaryotických hemových senzorových proteinů, které detegují plyny. Jedná se o přímý kyslíkový senzor z Escherichia coli, který vykazuje fosfodiesterasovou aktivitu (EcDOS) [54][55][56] , diguanylátcyklasu s globinovou strukturou senzorové domény z Escherichia coli (YddV) 42,55,57-59 a histidinkinasu s globinovou strukturou senzorové domény z půdní bakterie Anaeromyxobacter sp. kmen Fw109-5 (AfGcHK) 38,39,[60][61][62][63] .…”

A Zero-Valent Sulfur Transporter Helps Podophyllotoxin Uptake into Bacterial Cells in the Presence of CTAB

“…Sulfane sulfur is involved in many functions, including redox regulation of protein functions and cell redox homeostasis maintenance [24]. Sulfane sulfur species also act as signal molecules by modifying proteins at Cys residues or reacting with metal centers in proteins [25]. Sulfane sulfur may function as antioxidants as well as oxidants.…”