NO· Binds Human Cystathionine β-synthase Quickly and Tightly

Journal of Biological Chemistry

Nitric oxide (NO) synthases (NOSs) catalyze the formation of NO from L-arginine. We have shown previously that the NOS enzyme catalytic cycle involves a large number of reactions, but can be characterized by a global model with three main ratelimiting steps. These are the rate of heme reduction by the flavin domain (k r); of dissociation of NO from the ferric heme-NO complex (k d) and of oxidation of the ferrous heme-NO complex (k ox). The reaction of oxygen with the ferrous heme-NO species is part of a futile cycle that does not directly contribute to NO synthesis, but allows a population of inactive enzyme molecules to return to the catalytic cycle, and thus enables a steady-state NO synthesis rate. Previously, we have reported that this reaction does involve the reaction of oxygen with NO-bound ferrous heme complex, but the mechanistic details of the reaction, that could proceed via either an inner-sphere or an outer-sphere mechanism, remains unclear. Here we present additional experiments with neuronal NOS (nNOS) and inducible NOS (iNOS) variants (nNOS W409F and iNOS K82A and V346I) and computational methods to study how changes in heme access and electronics affect the reaction. Our results support an inner-sphere mechanism and indicate that the particular heme-thiolate environment of the NOS enzymes can stabilize an N-bound Fe III-N(O)OO − intermediate species and thereby catalyze this reaction, which otherwise is not observed or favorable in proteins like globins that contain a histidine-coordinated heme. _______________________________________ NO synthases (NOS, EC 1.14.13.39) are homodimeric enzymes that catalyze formation of the biological messenger molecule nitric oxide (NO) from L-arginine (L-arg) (1-3). Three main isoforms of NOS

Biochemistry, 2004

Human cystathionine-synthase (CBS) is a unique pyridoxal-5′-phosphate-dependent enzyme in which heme is also present as a cofactor. Because the function of heme in this enzyme has yet to be elucidated, the study presented herein investigated possible relationships between the chemistry of the heme and the strong pH dependence of CBS activity. This study revealed, via study of a truncation variant, that the catalytic core of the enzyme governs the pH dependence of the activity. The heme moiety was found to play no discernible role in regulating CBS enzyme activity by sensing changes in pH, because the coordination sphere of the heme is not altered by changes in pH over a range of pH 6-9. Instead, pH was found to control the equilibrium amount of ferric and ferrous heme present after reaction of CBS with one-electron reducing agents. A variety of spectroscopic techniques, including resonance Raman, magnetic circular dichroism, and electron paramagnetic resonance, demonstrated that at pH 9 Fe(II) CBS is dominant while at pH 6 Fe(III) CBS is favored. At low pH, Fe(II) CBS forms transiently but reoxidizes by an apparent proton-gated electron-transfer mechanism. Regulation of CBS activity by the iron redox state has been proposed as the role of the heme moiety in this enzyme. Given that the redox behavior of the CBS heme appears to be controlled by pH, interplay of pH and oxidation state effects must occur if CBS activity is redox regulated.

The Journal of biological chemistry, 2001

Nitric-oxide synthase (NOS) catalyzes the formation of NO and citrulline from l-arginine and oxygen. However, the NO so formed has been found to auto-inhibit the enzymatic activity significantly. We hypothesized that the NO reactivity is in part controlled by hydrogen bonding between the conserved tryptophan residue (position 409 in the neuronal isoform of NOS (nNOS)) and the cysteine residue that forms the proximal bond to the heme. By using resonance Raman spectroscopy and NO as a probe of the heme environment, we show that in the W409F and W409Y mutants of the oxygenase domain of the neuronal enzyme (nNOSox), the Fe-NO bond in the Fe3+NO complex is weaker than in the wild type enzyme, consistent with the loss of a hydrogen bond on the sulfur atom of the proximal cysteine residue. The weaker Fe-NO bond in the W409F and W409Y mutants might result in a faster rate of NO dissociation from the ferric heme in the Trp-409 mutants as compared with the wild type enzyme, which could contri...

Journal of the American Chemical Society, 2009

Cystathionine β-synthase (CBS) plays a central role in cysteine metabolism, and malfunction of the enzyme leads to homocystinuria, a devastating metabolic disease. CBS contains a pyridoxal 5′phosphate (PLP) cofactor which catalyzes the synthesis of cystathionine from homocysteine and serine. Mammalian forms of the enzyme also contain a heme group, which is not involved in catalysis. It may, however, play a regulatory role, since the enzyme is inhibited when CO or NO are bound to the heme. We have investigated the mechanism of this inhibition using fluorescence and resonance Raman spectroscopies. CO binding is found to induce a tautomeric shift of the PLP from the ketoenamine to the enolimine form. The ketoenamine is key to PLP reactivity because its imine C=N bond is protonated, facilitating attack by the nucleophilic substrate, serine. The same tautomer shift is also induced by heat inactivation of Fe(II)CBS, or by an Arg266Met replacement in Fe(II)CBS, which likewise inactivates the enzyme; in both cases the endogenous Cys52 ligand to the heme is replaced by another, unidentified ligand. CO binding also displaces Cys52 from the heme. We propose that the tautomer shift results from loss of a stabilizing H-bond from Asn149 to the PLP ring O4 atom, which is negatively charged in the ketoenamine tautomer. This loss would be induced by displacement of the PLP as a result of breaking the salt bridge between Cys52 and Arg266, which resides on a short helix that is also anchored to the PLP via H-bonds to its phosphate group. The salt bridge would be broken when Cys52 is displaced from the heme. Cys52 protonation is inferred to be the rate-limiting step in breaking the salt bridge, since the rate of the tautomer shift, following CO binding, increases with decreasing pH. In addition, elevation of the concentration of phosphate buffer was found to diminish the rate and extent of the tautomer shift, suggesting a ketoenamine-stabilizing phosphate binding site, possibly at the protonated imine bond of the PLP. Implications of these findings for CBS regulation are discussed.

Biochemistry, 1997

The substrate binding site in nitric oxide synthase (NOS) can accommodate the physiological substrates, L-arginine and N ω -hydroxy L-arginine as well as many substrate analogues and inhibitors. Resonance Raman spectra of carbon monoxide-bound NOS were measured to determine how these substrates and analogues interact with heme, the prosthetic group which activates oxygen for the catalytic generation of NO and citrulline from arginine in the enzyme. Two distinct conformations of the Fe-C-O moiety were detected in the resonance Raman spectra, although in the optical absorption spectra the two species are indistinguishable. In one, termed the -form, the Fe-CO stretching frequency and the C-O stretching frequency, located at ∼487 and ∼1949 cm -1 , respectively, demonstrate that the Fe-C-O group adopts a linear conformation perpendicular to the heme plane ("open" structure). In the other, termed the R-form, frequencies of ∼502 and ∼1929 cm -1 , respectively, indicate that the binding properties of bound CO are significantly affected by its immediate environment thereby leading to a "closed" structure. In the presence of L-arginine or N ω -OH-L-arginine all of the molecules exhibit the closed structure, indicating that the substrates exert a strong polar (and/or steric) effect on the hemebound ligand. In the absence of any substrate or inhibitor only half of the heme population adopts the open structure whereas the rest of the heme content retains the closed conformation. It is proposed that in this population with the closed structure tetrahydrobiopterin, a cofactor of NO synthase, may reside in close proximity to the heme-bound ligand and interact with it in a similar manner as do substrates. The inverse correlation between the Fe-CO and C-O stretching modes suggests that in NOS the bonding of the cysteine to the heme iron may be weaker, as found in chloroperoxidase, than in cytochrome P-450 enzymes. This work continually proves resonance Raman spectroscopy as a powerful probe for the interactions between substrate/inhibitor and the heme active site of proteins.

Antioxidants, 2021

The ‘gasotransmitters’ hydrogen sulfide (H2S), nitric oxide (NO), and carbon monoxide (CO) act as second messengers in human physiology, mediating signal transduction via interaction with or chemical modification of protein targets, thereby regulating processes such as neurotransmission, blood flow, immunomodulation, or energy metabolism. Due to their broad reactivity and potential toxicity, the biosynthesis and breakdown of H2S, NO, and CO are tightly regulated. Growing evidence highlights the active role of gasotransmitters in their mutual cross-regulation. In human physiology, the transsulfuration enzymes cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) are prominent H2S enzymatic sources. While CBS is known to be inhibited by NO and CO, little is known about CSE regulation by gasotransmitters. Herein, we investigated the effect of s-nitrosation on CSE catalytic activity. H2S production by recombinant human CSE was found to be inhibited by the physiological nitrosat...

Journal of the American Chemical Society, 2013

The synthesis and spectroscopic characterization of [Fe(NO)(N3PyS)]BF 4 (3) is presented, the first structural and electronic model of NO-bound cysteine dioxygenase (CDO). The nearly isostructural all-N-donor analog [Fe(NO)(N4Py)](BF 4 ) 2 (4) was also prepared, and comparisons of 3 and 4 provide insight regarding the influence of S versus N ligation in {FeNO} 7 species. One key difference occurs upon photoirradiation, which causes the fully reversible release of NO from 3, but not from 4. Mononuclear non-heme iron oxygenases utilize dioxygen to perform key oxidations in biology. Much effort has gone into obtaining a mechanistic understanding of these systems, including the trapping and characterization of metal-oxygen intermediates in both the proteins and synthetic models. The nitric oxide (NO•) molecule has been used as a surrogate for O 2 , generating Fe-NO adducts that are analogs for key Fe-O 2 intermediates and helping to determine the general site(s) and requirements for O 2 binding. Cysteine dioxygenase (CDO) is a non-heme iron oxygenase that is responsible for regulating cysteine levels in mammals by oxidizing cysteine to cysteine sulfinic acid with O 2 . The mechanism of CDO is still not well understood, although a number of experimental, computational, and synthetic studies have sought to address different features of the Soxygenation process. Pierce and co-workers employed NO• as an O 2 surrogate for CDO, and reported that, in the presence of Cys substrate, an iron-nitrosyl complex was formed (Fe(NO)(Cys)-CDO). 2c This {FeNO} 7 (Enemark-Feltham notation) 4 complex exhibited an unusual S = ½ ground state, as opposed to all other non-heme {FeNO} 7 enzymatic species, which exhibit an S = 3/2 ground state. A structural and functional model of CDO [Fe II (N3PyS)(CH 3 CN)]BF 4 (1), was previously described by some of us. 3a Addition of O 2 to 1 resulted in biomimetic S-oxygenation to give

Journal of Inorganic Biochemistry, 2003

Investigations on the biological effects of nitric oxide (NO) derived from nitric oxide synthase (NOS) have led to an explosion in biomedical research over the last decade. The chemistry of this diatomic radical is key to its biological effects. Recently, nitroxyl 2 (HNO / NO ) has been proposed to be another important constituent of NO biology. However, these redox siblings often exhibit orthogonal behavior in physiological and cellular responses. We therefore explored the chemistry of NO and HNO with heme proteins in different redox states and observed that HNO favors reaction with ferric heme while NO favors ferrous, consistent with previous reports. Further results show that HNO and NO were equally effective in inhibiting cytochrome P450 activity, which involves ferric and ferrous complexes. The differential chemical behavior of NO and HNO toward heme proteins provides insight into mechanisms of activity that not only helps explain some of the opposing effects observed in NOS-mediated events, but offers a unique control mechanism for the biological action of NO. Published by Elsevier Science Inc.

Biochemistry, 2002

Elevated levels of homocysteine, a sulfur-containing amino acid, are correlated with increased risk for cardiovascular diseases and Alzheimers disease and with neural tube defects. The only route for the catabolic removal of homocysteine in mammals begins with the pyridoxal phosphate-(PLP-) dependent-replacement reaction catalyzed by cystathionine-synthase. The enzyme has a b-type heme with unusual spectroscopic properties but as yet unknown function. The human enzyme has a modular organization and can be cleaved into an N-terminal catalytic core, which retains both the heme and PLP-binding sites and is highly active, and a C-terminal regulatory domain, where the allosteric activator S-adenosylmethionine is presumed to bind. Studies with the isolated recombinant enzyme and in transformed human liver cells indicate that the enzyme is ∼2-fold more active under oxidizing conditions. In addition to heme, the enzyme contains a CXXC oxidoreductase motif that could, in principle, be involved in redox sensing. In this study, we have examined the role of heme versus the vicinal thiols in modulating the redox responsiveness of the enzyme. Deletion of the heme domain leads to loss of redox sensitivity. In contrast, substitution of either cysteine with a non-redox-active amino acid does not affect the responsiveness of the enzyme to reductants. We also report the crystal structure of the catalytic core of the enzyme in which the vicinal cysteines are reduced without any discernible differences in the remainder of the protein. The structure of the catalytic core is compared to those of other members of the fold II family of PLPdependent enzymes and provides insights into active site residues that may be important in interacting with the substrates and intermediates.

Journal of Biological Chemistry, 2006

Cystathionine β-synthase (CBS), condenses homocysteine, a toxic metabolite, with serine, in a pyridoxal phosphate-dependent reaction. It also contains a heme cofactor, to which CO or NO can bind, resulting in enzyme inhibition. To understand the mechanism of this regulation, we have investigated the equilibria and kinetics of CO binding to the highly active catalytic core of CBS, which is dimeric. CBS exhibits strong anticooperativity in CO binding, with successive association constants of 0.24 and 0.02 μM −1 . Stopped-flow measurements reveal slow CO association (0.0166 s −1 ) limited by dissociation of the endogenous ligand, Cys52. Rebinding of CO and of Cys52 following CO photodissociation were independently monitored via time-resolved resonance Raman spectroscopy. The Cys52 rebinding rate, 4000 s −1 , is essentially unchanged between pH 7.6 and 10.5, indicating that the pK a of Cys52 is shifted below pH 7.6. This effect is attributed to the nearby Arg266 residue, which is proposed to form a salt-bridge with the dissociated Cys52, thereby inhibiting its protonation and slowing rebinding to the Fe. This salt-bridge suggests a pathway for enzyme inactivation upon CO binding, since Arg266 is located on a helix that connects the heme and pyridoxal phosphate cofactor domains.