Remains Containment Considerations for Death in Low-Earth Orbit.

BACKGROUND: Maintenance and disposition of decedent remains during spaceflight require the isolation of biohazardous products of decomposition in microgravity and in the absence of refrigeration. Containment and isolation options would preferably offer sufficient time to enable crew and ground support teams to determine appropriate disposition of remains and even potentially return remains to the Earth. The pilot study described herein undertook an effort to develop a postmortem containment unit for the isolation and maintenance of decedent remains in a microgravity environment.METHODS: Commercial off-the-shelf containment units were modified to meet the needs of a microgravity spaceflight environment and to offer the best likelihood of successful containment and management of remains. A subsequent evaluation of modified containment unit performance was undertaken utilizing human cadavers, with measurement and analysis of volatile off-gassing over time followed by impact testing of the units containing cadaverous remains in a simulated spaceflight vehicle seat.RESULTS: Modifications were implemented without significant negative design impact. Failure was observed in one modified unit after 9 d and attributed to improper filter application. The remaining unit successfully contained remains beyond the intended endpoint of the study.DISCUSSION: These pilot efforts offer important insight into the development of effective postmortem containment options for future spaceflight. Further study is needed to ensure repeatability of the findings and to further characterize the failure modes of the modified units evaluated, the impact of microgravity conditions, and the identification of additional modifications that would improve remains disposition.Houser T, Lindgren KN, Mazuchowski EL II, Barratt MR, Haines DC, Jayakody M, Blue RS, Bytheway JA, Stepaniak PC. Remains containment considerations for death in low-Earth orbit. Aerosp Med Hum Perform. 2023; 94(5):368-376.

Aluminum-substituted heme domain of P450BM-3 (BMP): introducing a heme-derived fluorescent probe for studies of substrate binding and protein-protein interactions in cytochromes P450.

We describe a novel technique for heme removal and replacement in the heme domain of P450BM-3 (BMP). The method was applied to obtain the aluminum-protoporphyrin IX (Al-PP) substituted derivative of BMP (Al-BMP). The overall yield of the purified Al-BMP was about 15% as related to the initial amount of the hemeprotein. Al-BMP possesses extensive fluorescence in the 550-650 nm region with excitation in the porphyrin absorbance bands. The protein was shown to bind substrates of P450BM-3 (palmitic, arachidonic, and cis-parinaric acids) with affinities similar to those of the native enzyme (3-6 µM). However, the substrate-induced changes in fluorescence of Al-PP reveal the existence of a second, low-affinity substrate-binding site, which cannot be detected by the spin shift in the native, heme-containing BMP. Using fluorescence resonance energy transfer, we have demonstrated that Al-BMP forms a complex with the flavoprotein domain of P450BM-3 labeled with 7-ethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin maleimide, revealing the affinity similar to that of native BMP (Kd = 5 µM at 0.06 M ionic strength). Therefore, aluminum-substituted BMP may serve as a valuable tool in studies on the mechanisms of interactions of P450s with their substrates and protein partners.

Comparison of brain mitochondrial cytochrome c oxidase activity with cyanide LD(50) yields insight into the efficacy of prophylactics.

Cyanide inhibits cytochrome c oxidase, the terminal oxidase of the mitochondrial respiratory pathway, therefore inhibiting the cell oxygen utilization and resulting in the condition of histotoxic anoxia. The enzyme rhodanese detoxifies cyanide by utilizing sulfur donors to convert cyanide to thiocyanate, and new and improved sulfur donors are actively sought as researchers seek to improve cyanide prophylactics. We have determined brain cytochrome c oxidase activity as a marker for cyanide exposure for mice pre-treated with various cyanide poisoning prophylactics, including sulfur donors thiosulfate (TS) and thiotaurine (TT3). Brain mitochondria were isolated by differential centrifugation, the outer mitochondrial membrane was disrupted by a maltoside detergent, and the decrease in absorbance at 550 nm as horse heart ferrocytochrome c (generated by the dithiothreitol reduction of ferricytochrome c) was oxidized was monitored. Overall, the TS control prophylactic treatment provided significant protection of the cytochrome c oxidase activity. The TT3-treated mice showed reduced cytochrome c oxidase activity even in the absence of cyanide. In both treatment series, addition of exogenous Rh did not significantly enhance the prevention of cytochrome c oxidase inhibition, but the addition of sodium nitrite did. These findings can lead to a better understanding of the protection mechanism by various cyanide antidotal systems.

Peroxidase-like activity of uncoupled cytochrome P450: studies with bilirubin and toxicological implications of uncoupling.

The NADPH-dependent consumption of O(2) by cytochrome P450 BM3 was stimulated by either laurate or perfluorolaurate, but the NADPH/O(2) molar consumption ratios were approximately 1 and 2, respectively, indicating that perfluorolaurate does not become oxygenated by BM3 and oxygen undergoes full reduction to water. The nature of this catalytic cycle uncoupled to hydroxylation was explored using bilirubin as a molecular probe. During uncoupling with perfluorolaurate bilirubin was degraded and stimulated O(2) uptake by an approximately equimolar amount. No stimulation of oxygen uptake was caused by bilirubin in presence of NADPH alone or in presence of laurate together with NADPH; under these conditions little degradation of bilirubin was observed. Mesobilirubin was also degraded during uncoupling with perfluorolaurate, whereas biliverdin (which lacks the central methene bridge present in rubins) was unaffected. It is suggested that the CYP ferryl oxygen species abstracts a hydrogen atom from the central methene bridge of bilirubin to generate a radical, which is further dehydrogenated to biliverdin or else binds O(2) and undergoes fragmentation. We conclude that the uncoupled catalytic cycle of cytochrome P450 has properties resembling those of a peroxidase and that bilirubin is rapidly oxidized as a peroxidase substrate. The potential toxicological significance of cytochrome P450 uncoupling is considered.

A single active-site mutation of P450BM-3 dramatically enhances substrate binding and rate of product formation.

Identifying key structural features of cytochromes P450 is critical in understanding the catalytic mechanism of these important drug-metabolizing enzymes. Cytochrome P450BM-3 (BM-3), a structural and mechanistic P450 model, catalyzes the regio- and stereoselective hydroxylation of fatty acids. Recent work has demonstrated the importance of water in the mechanism of BM-3, and site-specific mutagenesis has helped to elucidate mechanisms of substrate recognition, binding, and product formation. One of the amino acids identified as playing a key role in the active site of BM-3 is alanine 328, which is located in the loop between the K helix and ß 1-4. In the A328V BM-3 mutant, substrate affinity increases 5-10-fold and the turnover number increases 2-8-fold compared to wild-type enzyme. Unlike wild-type enzyme, this mutant is purified from E. coli with endogenous substrate bound due to the higher binding affinity. Close examination of the crystal structures of the substrate-bound native and A328V mutant BMPs indicates that the positioning of the substrate is essentially identical in the two forms of the enzyme, with the two valine methyl groups occupying voids present in the active site of the wild-type substrate-bound structure.

Paraoxonase 2 is down-regulated by the Pseudomonas aeruginosa quorumsensing signal N-(3-oxododecanoyl)-L-homoserine lactone and attenuates oxidative stress induced by pyocyanin.

Two virulence factors produced by Pseudomonas aeruginosa are pyocyanin and N-(3-oxododecanoyl)-L-homoserine lactone (3OC12). Pyocyanin damages host cells by generating ROS (reactive oxygen species). 3OC12 is a quorum-sensing signalling molecule which regulates bacterial gene expression and modulates host immune responses. PON2 (paraoxonase-2) is an esterase that inactivates 3OC12 and potentially attenuates Ps. aeruginosa virulence. Because increased intracellular Ca2+ initiates the degradation of PON2 mRNA and protein and 3OC12 causes increases in cytosolic Ca2+, we hypothesized that 3OC12 would also down-regulate PON2. 3OC12 and the Ca2+ ionophore A23187 caused a rapid cytosolic Ca2+ influx and down-regulated PON2 mRNA, protein and hydrolytic activity in A549 and EA.hy 926 cells. The decrease in PON2 hydrolytic activity was much more extensive and rapid than decreases in protein, suggesting a rapid post-translational mechanism which blocks PON2's hydrolytic activity. The Ca2+ chelator BAPTA/AM [1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester)] diminished the ability of 3OC12 to decrease PON2, demonstrating that the effects are mediated by Ca2+. PON2 also has antioxidative properties and we show that it protects cells from pyocyanin-induced oxidative stress. Knockdown of PON2 by transfecting cells with siRNA (small interfering RNA) rendered them more sensitive to, whereas overexpression of PON2 protected cells from, pyocyanin-induced ROS formation. Additionally, 3OC12 potentiated pyocyanin-induced ROS formation, presumably by inactivating PON2. These findings support a key role for PON2 in the defence against Ps. aeruginosa virulence, but also reveal a mechanism by which the bacterium may subvert the protection afforded by PON2.

A single mutation in P450BM-3 enhances acyl homoserine lactone: acyl homoserine substrate binding selectivity nearly 250-fold.

Quorum sensing is the process by which bacteria alter gene regulation in response to their population density. The enzymatic inactivation of quorum signals has shown promise for use in genetically modified organisms resistant to pathogens. We recently characterized the ability of a cytochrome P450, P450BM-3, to oxidize the quorum sensing signals known as acyl homoserine lactones. The oxidation of the acyl homoserine lactones reduced their activity as quorum signals. The enzyme also oxidized the inactive lactonolysis products, acyl homoserines. The enzyme showed similar binding affinity for the acyl homoserine lactones and acyl homoserines. The latter reaction may lead to problems when lactonases and the P450-dependent system are used in tandem, as oxidation of the acyl homoserines produced by lactonolysis in vivo may compete with acyl homoserine lactone oxidation by the cytochrome P450. We report here that a single mutation (R47S) in P450BM-3 is capable of increasing the acyl homoserine lactone: acyl homoserine substrate binding selectivity of the enzyme nearly 250-fold, reducing the potential for competition by acyl homoserines and significantly enhancing the potential for use of P450BM-3 as part of a pathogen resistance system in genetically modified crops.

Dominant role of paraoxonases in inactivation of the Pseudomonas aeruginosa quorum-sensing signal N-(3-oxododecanoyl)-L-homoserine lactone.

The pathogenic bacterium Pseudomonas aeruginosa causes serious infections in immunocompromised patients. N-(3-oxododecanoyl)-L-homoserine lactone (3OC12-HSL) is a key component of P. aeruginosa's quorum-sensing system and regulates the expression of many virulence factors. 3OC12-HSL was previously shown to be hydrolytically inactivated by the paraoxonase (PON) family of calcium-dependent esterases, consisting of PON1, PON2, and PON3. Here we determined the specific activities of purified human PONs for 3OC12-HSL hydrolysis, including the common PON1 polymorphic forms, and found they were in the following order: PON2 >> PON1(192R) > PON1(192Q) > PON3. PON2 exhibited a high specific activity of 7.6 +/- 0.4 micromols/min/mg at 10 microM 3OC12-HSL, making it the best PON2 substrate identified to date. By use of class-specific inhibitors, approximately 85 and 95% of the 3OC12-HSL lactonase activity were attributable to PON1 in mouse and human sera, respectively. In mouse liver homogenates, the activity was metal dependent, with magnesium- and manganese-dependent lactonase activities comprising 10 to 15% of the calcium-dependent activity. In mouse lung homogenates, all of the activity was calcium dependent. The calcium-dependent activities were irreversibly inhibited by extended EDTA treatment, implicating PONs as the major enzymes inactivating 3OC12-HSL. In human HepG2 and EA.hy 926 cell lysates, the 3OC12-HSL lactonase activity closely paralleled the PON2 protein levels after PON2 knockdown by small interfering RNA treatment of the cells. These findings suggest that PONs, particularly PON2, could be an important mechanism by which 3OC12-HSL is inactivated in mammals.

Crystal structure of inhibitor-bound P450BM-3 reveals open conformation of substrate access channel.

P450BM-3 is an extensively studied P450 cytochrome that is naturally fused to a cytochrome P450 reductase domain. Crystal structures of the heme domain of this enzyme have previously generated many insights into features of P450 structure, substrate binding specificity, and conformational changes that occur on substrate binding. Although many P450s are inhibited by imidazole, this compound does not effectively inhibit P450BM-3. Omega-imidazolyl fatty acids have previously been found to be weak inhibitors of the enzyme and show some unusual cooperativity with the substrate lauric acid. We set out to improve the properties of these inhibitors by attaching the omega-imidazolyl fatty acid to the nitrogen of an amino acid group, a tactic that we used previously to increase the potency of substrates. The resulting inhibitors were significantly more potent than their parent compounds lacking the amino acid group. A crystal structure of one of the new inhibitors bound to the heme domain of P450BM-3 reveals that the mode of interaction of the amino acid group with the enzyme is different from that previously observed for acyl amino acid substrates. Further, required movements of residues in the active site to accommodate the imidazole group provide an explanation for the low affinity of imidazole itself. Finally, the previously observed cooperativity with lauric acid is explained by a surprisingly open substrate-access channel lined with hydrophobic residues that could potentially accommodate lauric acid in addition to the inhibitor itself.

Interactions of substrates at the surface of P450s can greatly enhance substrate potency.

Cytochrome P450s are a superfamily of heme containing enzymes that use molecular oxygen and electrons from reduced nicotinamide cofactors to monooxygenate organic substrates. The fatty acid hydroxylase P450BM-3 has been particularly widely studied due to its stability, high activity, similarity to mammalian P450s, and presence of a cytochrome P450 reductase domain that allows the enzyme to directly receive electrons from NADPH without a requirement for additional redox proteins. We previously characterized the substrate N-palmitoylglycine, which found extensive use in studies of P450BM-3 due to its high affinity, high turnover number, and increased solubility as compared to fatty acid substrates. Here, we report that even higher affinity substrates can be designed by acylation of other amino acids, resulting in P450BM-3 substrates with dissociation constants below 100 nM. N-Palmitoyl-l-leucine and N-palmitoyl-l-methionine were found to have the highest affinity, with dissociation constants of less than 8 nM and turnover numbers similar to palmitic acid and N-palmitoylglycine. The interactions of the amino acid side chains with a hydrophobic pocket near R47, as revealed by our crystal structure determination of N-palmitoyl-l-methionine bound to the heme domain of P450BM-3, appears to be responsible for increasing the affinity of substrates. The side chain of R47, previously shown to be important in interactions with negatively charged substrates, does not interact strongly with N-palmitoyl-l-methionine and is found positioned at the enzyme-solvent interface. These are the tightest binding substrates for P450BM-3 reported to date, and the affinity likely approaches the maximum attainable affinity for the binding of substrates of this size to P450BM-3.

Bacillus megaterium CYP102A1 oxidation of acyl homoserine lactones and acyl homoserines.

Quorum sensing, the ability of bacteria to sense their own population density through the synthesis and detection of small molecule signals, has received a great deal of attention in recent years. Acyl homoserine lactones (AHLs) are a major class of quorum sensing signaling molecules. In nature, some bacteria that do not synthesize AHLs themselves have developed the ability to degrade these compounds by cleaving the amide bond or the lactone ring. By inactivating this signal used by competing bacteria, the degrading microbe is believed to gain a competitive advantage. In this work we report that CYP102A1, a widely studied cytochrome P450 from Bacillus megaterium, is capable of very efficient oxidation of AHLs and their lactonolysis products acyl homoserines. The previously known substrates for this enzyme, fatty acids, can also be formed in nature by hydrolysis of the amide of AHLs, so CYP102A1 is capable of inactivating the active parent compound and the products of both known pathways for AHL inactivation observed in nature. AHL oxidation primarily takes place at the omega-1, omega-2, and omega-3 carbons of the acyl chain, similar to this enzyme's well-known activity on fatty acids. Acyl homoserines and their lactones are better substrates for CYP102A1 than fatty acids. Bioassay of the quorum sensing activity of oxidation products reveals that the subterminally hydroxylated AHLs exhibit quorum sensing activity, but are 18-fold less active than the parent compound. In vivo, B. megaterium inactivates AHLs by a CYP102A1 dependent mechanism that must involve additional components that further sequester or metabolize the products, eliminating their quorum sensing activity. Cytochrome P450 oxidation of AHLs represents an important new mechanism of quorum quenching.

Cloning, expression and characterization of a fast self-sufficient P450: CYP102A5 from Bacillus cereus.

CYP102s represent a family of natural self-sufficient fusions of cytochrome P450 and cytochrome P450 reductase found in some bacteria. One member of this family, named CYP102A1 or more traditionally P450BM-3, has been widely studied as a model of human P450 cytochromes. Remarkable detail of P450 structure and function has been revealed using this highly efficient enzyme. The recent rapid expansion of microbial genome sequences has revealed many relatives of CYP102A1, but to date only two from Bacillus subtilis have been characterized. We report here the cloning and expression of CYP102A5, a new member of this family that is very closely related to CYP102A4 from Bacillus anthracis. Characterization of the substrate specificity of CYP102A5 shows that it, like the other CYP102s, will metabolize saturated and unsaturated fatty acids as well as N-acylamino acids. CYP102A5 catalyzes very fast substrate oxidation, showing one of the highest turnover rates for any P450 monooxygenase studied so far. It does so with more specificity than other CYP102s, yielding primarily omega-1 and omega-2 hydroxylated products. Measurement of the rate of electron transfer through the reductase domain reveals that it is significantly faster in CYP102A5 than in CYP102A1, providing a likely explanation for the increased monooxygenation rate. The availability of this new, very fast fusion P450 will provide a great tool for comparative structure-function studies between CYP102A5 and the other characterized CYP102s.

Obligatory intermolecular electron-transfer from FAD to FMN in dimeric P450BM-3.

Cytochromes P450 typically catalyze the monooxygenation of hydrophobic compounds resulting in the insertion of one atom of dioxygen into the organic substrate and the reduction of the other oxygen atom to water. The two electrons required for the reaction are normally provided by another redox active protein, for example cytochrome P450 reductase (CPR) in mammalian endoplasmic reticulum membranes. P450BM-3 from Bacillus megaterium is a widely studied P450 cytochrome in which the P450 is fused naturally to a diflavin reductase homologous to CPR. From the original characterization of the enzyme by Fulco's laboratory, the enzyme was shown to have a nonlinear dependence of reaction rate on enzyme concentration. In recent experiments we observed enzyme inactivation upon dilution, and the presence of substrate can diminish this inactivation. We therefore carried out enzyme kinetics, cross-linking experiments, and molecular weight determinations that establish that the enzyme is capable of dimerizing in solution. The dimer is the predominant form at higher concentrations under most conditions and is the only form with significant activity. Further experiments selectively knocking out the activity of individual domains with site-directed mutagenesis and measuring enzyme activity in heterologous dimers establish that the electron-transfer pathway in P450BM-3 passes through both protein molecules in the dimer during a single turnover, traversing from the FAD domain of one molecule into the FMN domain of the other molecule before passing to the heme domain. Analysis of our results combined with other analyses in the literature suggests that the heme domain of either monomer may accept electrons from the reduced FMN domain.

A role for the strained phenylalanine ring rotation induced by substrate binding to cytochrome CYP102A1.

X-ray crystal structures of CYP102A1 (P450BM-3) have shown that PHE87 rotates upon substrate binding and is in contact with the heme cofactor. Analysis of substrate binding data combined with DFT calculations suggest that the ring is rotated into an unfavorable interaction with the heme and this could drive active site rearrangement.

L-Canavanine made by Medicago sativa interferes with quorum sensing in Sinorhizobium meliloti.

Sinorhizobium meliloti is a gram-negative soil bacterium, capable of establishing a nitrogen-fixing symbiosis with its legume host, alfalfa (Medicago sativa). Quorum sensing plays a crucial role in this symbiosis, where it influences the nodulation process and the synthesis of the symbiotically important exopolysaccharide II (EPS II). S. meliloti has three quorum-sensing systems (Sin, Tra, and Mel) that use N-acyl homoserine lactones as their quorum-sensing signal molecule. Increasing evidence indicates that certain eukaryotic hosts involved in symbiotic or pathogenic relationships with gram-negative bacteria produce quorum-sensing-interfering (QSI) compounds that can cross-communicate with the bacterial quorum-sensing system. Our studies of alfalfa seed exudates suggested the presence of multiple signal molecules capable of interfering with quorum-sensing-regulated gene expression in different bacterial strains. In this work, we choose one of these QSI molecules (SWI) for further characterization. SWI inhibited violacein production, a phenotype that is regulated by quorum sensing in Chromobacterium violaceum. In addition, this signal molecule also inhibits the expression of the S. meliloti exp genes, responsible for the production of EPS II, a quorum-sensing-regulated phenotype. We identified this molecule as l-canavanine, an arginine analog, produced in large quantities by alfalfa and other legumes.

Modulation of zinc- and cobalt-binding affinities through changes in the stability of the zinc ribbon protein L36.

Cysteine-rich Zn(II)-binding sites in proteins serve two distinct functions: to template or stabilize specific protein folds, and to facilitate chemical reactions such as alkyl transfers. We are interested how the protein environment controls metal site properties, specifically, how naturally occurring tetrahedral Zn(II) sites are affected by the surrounding protein. We have studied the Co(II)- and Zn(II)-binding of a series of derivatives of L36, a small zinc ribbon protein containing a (Cys)(3)His metal coordination site. UV-vis spectroscopy was used to monitor metal binding by peptides at pH 6.0. For all derivatives, the following trends were observed: (1) Zn(II) binds tighter than Co(II), with an average K (A) (Zn) /K (A) (Co) of 2.8(+/-2.0)x10(3); (2) mutation of the metal-binding ligand His32 to Cys decreases the affinity of L36 derivatives for both metals; (3) a Tyr24 to Trp mutation in the beta-sheet hydrophobic cluster increases K (A) (Zn) and K (A) (Co) ; (4) mutation in the beta-hairpin turn, His20 to Asn generating an Asn-Gly turn, also increases K (A) (Zn) and K (A) (Co) ; (5) the combination of His20 to Asn and Tyr24 to Trp mutations also increases K (A) (Zn) and K (A) (Co) , but the increments versus C(3)H are less than those of the single mutations. Furthermore, circular dichroism, size-exclusion chromatography, and 1D and 2D (1)H NMR experiments show that the mutations do not change the overall fold or association state of the proteins. L36, displaying Co(II)- and Zn(II)-binding sensitivity to various sequence mutations without undergoing a change in protein structure, can therefore serve as a useful model system for future structure/reactivity studies.

pH-induced alteration and oxidative destruction of heme in purified chromaffin granule cytochrome b(561): implications for the oxidative stress in catecholaminergic neurons.

The transmembrane hemoprotein, cytochrome b(561) (b(561)), in the neuroendocrine secretory vesicles is shown to shuttle electrons from the cytosolic ascorbate (Asc) to the intravesicular matrix to provide reducing equivalents for the dopamine beta-monooxygenase (DbetaM) reaction. Intravesicular Asc may also play a role in relieving catecholamine-induced oxidative stress in catecholaminergic neurons. In the present study, we have examined the alteration of purified oxidized b(561) (b(561,ox)) under mild alkaline conditions to probe the structural and functional characteristics of the protein, using UV-vis and EPR spectroscopic and kinetic techniques. Our results show that low spin heme in oxidized b(561) (b(561,ox)) readily transforms to an altered high spin form and then slowly to an Asc nonreducible form, in a pH-, temperature-, and time-dependent manner, which can be described by single-exponential rate equations, A(t) = A(o)(1- e (-kt)) and A(t) = A(o)e(-kt), respectively. More than half of the Asc nonreducible altered b(561) could be converted back to the native b(561) by pH adjustment followed by dithionite reduction, suggesting the reversibility of the process. The heme center of the transformed Asc nonreducible protein is completely bleached instantaneously by dithionite in the presence of atmospheric oxygen, which appears to be mediated by molecular oxygen and/or hydrogen peroxide. These results demonstrate that the heme centers of the protein are susceptible to the pH-induced alteration and oxidative destruction, raising some questions regarding the proposed one alkaline labile, two-heme model of b(561) [Tsubaki, M.; Nakayama, M.; Okuyama, E.; Ichikawa, Y. (1997) J. Biol. Chem. 272, 23206-23210]. The pH-induced alteration and the destruction of heme under oxidative conditions may play a significant role in the amplification of oxidative stress in catecholaminergic neurons.

Plausible molecular mechanism for activation by fumarate and electron transfer of the dopamine beta-mono-oxygenase reaction.

A series of fumarate analogues has been used to explore the molecular mechanism of the activation of dopamine beta-mono-oxygenase by fumarate. Mesaconic acid (MA) and trans -glutaconic acid (TGA) both activate the enzyme at low concentrations, similar to fumarate. However, unlike fumarate, TGA and MA interact effectively with the oxidized enzyme to inhibit it at concentrations of 1-5 mM. Monoethylfumarate (EFum) does not activate the enzyme, but inhibits it. In contrast with TGA and MA, however, EFum inhibits the enzyme by interacting with the reduced form. The saturated dicarboxylic acid analogues, the geometric isomer and the diamide of fumaric acid do not either activate or inhibit the enzyme. The phenylethylamine-fumarate conjugate, N -(2-phenylethyl)fumaramide (PEA-Fum), is an approximately 600-fold more potent inhibitor than EFum and behaves as a bi-substrate inhibitor for the reduced enzyme. The amide of PEA-Fum behaves similarly, but with an inhibition potency approximately 20-fold less than that of PEA-Fum. The phenylethylamine conjugates of saturated or geometric isomers of fumarate do not inhibit the enzyme. Based on these findings and on steady-state kinetic analysis, an electrostatic model involving an interaction between the amine group of the enzyme-bound substrate and a carboxylate group of fumarate is proposed to account for enzyme activation by fumarate. Furthermore, in light of the recently proposed model for the similar copper enzyme, peptidylglycine alpha-hydroxylating mono-oxygenase, the above electrostatic model suggests that fumarate may also play a role in efficient electron transfer between the active-site copper centres of dopamine beta-mono-oxygenase.