The History of Animal and Plant Sulfite Oxidase-A Personal View.

Sulfite oxidase is one of five molybdenum-containing enzymes known in eukaryotes where it catalyzes the oxidation of sulfite to sulfate. This review covers the history of sulfite oxidase research starting out with the early years of its discovery as a hepatic mitochondrial enzyme in vertebrates, leading to basic biochemical and structural properties that have inspired research for decades. A personal view on sulfite oxidase in plants, that sulfates are assimilated for their de novo synthesis of cysteine, is presented by Ralf Mendel with numerous unexpected findings and unique properties of this single-cofactor sulfite oxidase localized to peroxisomes. Guenter Schwarz connects his research to sulfite oxidase via its deficiency in humans, demonstrating its unique role amongst all molybdenum enzymes in humans. In essence, in both the plant and animal kingdoms, sulfite oxidase represents an important player in redox regulation, signaling and metabolism, thereby connecting sulfur and nitrogen metabolism in multiple ways.

Automated Image Analysis Reveals Different Localization of Synaptic Gephyrin C4 Splice Variants.

Postsynaptic scaffolding proteins function as central organization hubs, ensuring the synaptic localization of neurotransmitter receptors, trans-synaptic adhesion proteins, and signaling molecules. Gephyrin is the major postsynaptic scaffolding protein at glycinergic and a subset of GABAergic inhibitory synapses. In contrast to cells outside the CNS, where one gephyrin isoform is predominantly expressed, neurons express different splice variants. In this study, we characterized the expression and scaffolding of neuronal gephyrin isoforms differing in the inclusion of the C4 cassettes located in the central C-domain. In hippocampal and cortical neuronal populations, gephyrin P1, lacking additional cassettes, is the most abundantly expressed isoform. In addition, alternative splicing generated isoforms carrying predominantly C4a, and minor amounts of C4c or C4d cassettes. We detected no striking difference in C4 isoform expression between different neuron types and a single neuron can likely express all C4 isoforms. To avoid the cytosolic aggregates that are commonly observed upon exogenous gephyrin expression, we used adeno-associated virus (AAV)-mediated expression to analyze the scaffolding behavior of individual C4 isoforms in murine dissociated hippocampal glutamatergic neurons. While all isoforms showed similar clustering at GABAergic synapses, a thorough quantitative analysis revealed localization differences for the C4c isoform (also known as P2). Specifically, synaptic C4c isoform clusters showed a more distal dendritic localization and reduced occurrence at P1-predominating synapses. Additionally, inhibitory currents displayed faster decay kinetics in the presence of gephyrin C4c compared with P1. Therefore, inhibitory synapse heterogeneity may be influenced, at least in part, by mechanisms relating to C4 cassette splicing.

Molybdenum Cofactor Deficiency in Humans.

Molybdenum cofactor (Moco) deficiency (MoCD) is characterized by neonatal-onset myoclonic epileptic encephalopathy and dystonia with cerebral MRI changes similar to hypoxic-ischemic lesions. The molecular cause of the disease is the loss of sulfite oxidase (SOX) activity, one of four Moco-dependent enzymes in men. Accumulating toxic sulfite causes a secondary increase of metabolites such as S-sulfocysteine and thiosulfate as well as a decrease in cysteine and its oxidized form, cystine. Moco is synthesized by a three-step biosynthetic pathway that involves the gene products of MOCS1, MOCS2, MOCS3, and GPHN. Depending on which synthetic step is impaired, MoCD is classified as type A, B, or C. This distinction is relevant for patient management because the metabolic block in MoCD type A can be circumvented by administering cyclic pyranopterin monophosphate (cPMP). Substitution therapy with cPMP is highly effective in reducing sulfite toxicity and restoring biochemical homeostasis, while the clinical outcome critically depends on the degree of brain injury prior to the start of treatment. In the absence of a specific treatment for MoCD type B/C and SOX deficiency, we summarize recent progress in our understanding of the underlying metabolic changes in cysteine homeostasis and propose novel therapeutic interventions to circumvent those pathological changes.

A systematic analysis of diet-induced nephroprotection reveals overlapping changes in cysteine catabolism.

Caloric Restriction (CR) extends lifespan and augments cellular stress-resistance from yeast to primates, making CR an attractive strategy for organ protection in the clinic. Translation of CR to patients is complex, due to problems regarding adherence, feasibility, and safety concerns in frail patients. Novel tailored dietary regimens, which modulate the dietary composition of macro- and micronutrients rather than reducing calorie intake promise similar protective effects and increased translatability. However, a direct head-to-head comparison to identify the most potent approach for organ protection, as well as overlapping metabolic consequences have not been performed. We systematically analyzed six dietary preconditioning protocols - fasting mimicking diet (FMD), ketogenic diet (KD), dietary restriction of branched chained amino acids (BCAA), two dietary regimens restricting sulfur-containing amino acids (SR80/100) and CR - in a rodent model of renal ischemia-reperfusion injury (IRI) to quantify diet-induced resilience in kidneys. Of the administered diets, FMD, SR80/100 and CR efficiently protect from kidney damage after IRI. Interestingly, these approaches show overlapping changes in oxidative and hydrogen sulfide (H2S)-dependent cysteine catabolism as a potential common mechanism of organ protection.

[Perioperative hypothermia in dogs receiving combined administration of acepromazin and metamizol]. / Untersuchung der perioperativen Hypothermie bei kombinierter Gabe von Acepromazin und Metamizol beim Hund.

OBJECTIVE: Evaluation the development of perioperative body temperature while administrating a combination of acepromazine and metamizol (AM) versus anesthesia with acepromazine (A) alone. MATERIAL AND METHODS: In this prospective, quasi-randomized controlled study 20 dogs undergoing standardized tibial plateau leveling osteotomy were alternatingly assigned to group A or group AM (n = 10 each). The patients' body temperature values were recorded from the time of premedication up to its post-surgical return to reference values. RESULTS: Body temperature decreases during anesthesia in both groups were comparable (p = 0.12). Postoperatively on the other hand, temperature development differed significantly between the two groups (p = 0.0455). In 6 dogs of the group AM, body temperature continued to decrease following extubation prior to returning to normothermic values. CONCLUSION: Intraoperatively, all patients developed hypothermia, regardless of the investigated anesthetic medication administered. Postoperatively, patients not receiving metamizol reached normothermia more rapidly.

Short-Term Clinical and Radiographical Outcome after Application of Anchored Intervertebral Spacers in Dogs with Disc-Associated Cervical Spondylomyelopathy.

OBJECTIVES: The purpose of this study was to assess the short-term outcome of a new intervertebral anchored fusion device (C-LOX) for the treatment of disc associated cervical spondylomyelopathy (DA-CSM) in dogs, based on clinical and radiographical follow-up data. MATERIALS AND METHODS: To be included in the study, dogs had to be clinically affected by DA-CSM treated with surgical distraction/stabilization using the anchored intervertebral spacer (C-LOX). Neurological signs, as well as diagnostic imaging performed pre-, immediately postoperatively, and after 6 weeks and 3 months were assessed. If available, clinical follow-up after 3 months was documented. RESULTS: Thirty-seven cases were enrolled in the study. Outcome at 3 months was available in 25 dogs; improvement of neurological status was documented in 25/25 cases.The most common postoperative complication was screw loosening and/or breakage (n = 22), followed by subsidence (n = 15). Four dogs required revision surgery. CLINICAL SIGNIFICANCE: Distraction/stabilization of DA-CSM in dogs with the C-LOX device resulted in short-term clinical improvement in 33/37 treated cases. The high incidence of screw loosening was taken into consideration and modification of the implant with a new locking system and new screw dimensions was required. The C-LOX device seems to be a valuable alternative to more complicated distraction-fusion techniques.

Highlighted mechanistic aspects in the chemical biology of reactive sulfur species.

LINKED ARTICLES: This article is part of a themed section on Chemical Biology of Reactive Sulfur Species. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.4/issuetoc.

Molecular cloning, expression and biochemical characterization of periplasmic nitrate reductase from Campylobacter jejuni.

Campylobacter jejuni, a human gastrointestinal pathogen, uses nitrate for growth under microaerophilic conditions using periplasmic nitrate reductase (Nap). The catalytic subunit, NapA, contains two prosthetic groups, an iron sulfur cluster and a molybdenum cofactor. Here we describe the cloning, expression, purification, and Michaelis-Menten kinetics (kcat of 5.91 ± 0.18 s-1 and a KM (nitrate) of 3.40 ± 0.44 µM) in solution using methyl viologen as an electron donor. The data suggest that the high affinity of NapA for nitrate could support growth of C. jejuni on nitrate in the gastrointestinal tract. Site-directed mutagenesis was used and the codon for the molybdenum coordinating cysteine residue has been exchanged for serine. The resulting variant NapA is 4-fold less active than the native enzyme confirming the importance of this residue. The properties of the C. jejuni enzyme reported here represent the first isolation and characterization of an epsilonproteobacterial NapA. Therefore, the fundamental knowledge of Nap has been expanded.

A mild case of molybdenum cofactor deficiency defines an alternative route of MOCS1 protein maturation.

Molybdenum cofactor deficiency is an autosomal recessive inborn error of metabolism, which results from mutations in genes involved in Moco biosynthesis. Moco serves as a cofactor of several enzymes, including sulfite oxidase. MoCD is clinically characterized by intractable seizures and severe, rapidly progressing neurodegeneration leading to death in early childhood in the majority of known cases. Here we report a patient with an unusual late disease onset and mild phenotype, characterized by a lack of seizures, normal early development, a decline triggered by febrile illness and a subsequent dystonic movement disorder. Genetic analysis revealed a homozygous c.1338delG MOCS1 mutation causing a frameshift (p.S442fs) with a premature termination of the MOCS1AB translation product at position 477 lacking the entire MOCS1B domain. Surprisingly, urine analysis detected trace amounts (1% of control) of the Moco degradation product urothione, suggesting a residual Moco synthesis in the patient, which was consistent with the mild clinical presentation. Therefore, we performed bioinformatic analysis of the patient's mutated MOCS1 transcript and found a potential Kozak-sequence downstream of the mutation site providing the possibility of an independent expression of a MOCS1B protein. Following the expression of the patient's MOCS1 cDNA in HEK293 cells we detected two proteins: a truncated MOCS1AB protein and a 22.4 kDa protein representing MOCS1B. Functional studies of both proteins confirmed activity of MOCS1B, but not of the truncated MOCS1AB. This finding demonstrates an unusual mechanism of translation re-initiation in the MOCS1 transcript, which results in trace amounts of functional MOCS1B protein being sufficient to partially protect the patient from the most severe symptoms of MoCD.

Slipped capital femoral epiphysis in 17 Maine Coon cats.

Objectives From May 2009 to January 2015, 208 Maine Coon cats presented to the Tierklinik Hollabrunn - a small animal referral and first-opinion centre - and 17 (8.17%) cats were diagnosed with a slipped capital femoral epiphysis (SCFE). Over the same time period, 29 (0.67%) of 4348 cats (all breeds) were diagnosed with SCFE. Methods Clinical and orthopaedic examinations and diagnostic imaging were performed on all affected Maine Coons. Age at first presentation, sex, body weight, body condition score (BCS), unilateral or bilateral manifestation of the disease, activity level and duration of lameness, age at neutering and known family history of disease were recorded. Sixteen of 17 Maine Coons were surgically treated. Surgically removed femoral tissue samples were histologically examined in 13 cases. Results The mean age at first presentation was 21.47 months; male to female ratio was 16:1; mean body weight was 7.5 kg (range 5.3-9.3 kg); and mean BCS was 5.06/9.0. Seven cats were bilaterally affected; the median duration of decreased activity level and lameness was 2 weeks; mean age at neutering was 7.7 months (range 3.0-12.0 months); and four cats were littermates. Fourteen femoral head and neck ostectomies, eight total hip replacements and one primary fixation were performed. All 13 histologically available samples confirmed the diagnosis of SCFE. Conclusions and relevance To date, SCFE has been reported only occasionally in Maine Coon cats. However, the results of this study showed that Maine Coons were approximately 12-fold more likely to develop SCFE than the overall population of cats presenting to the Tierklinik Hollabrunn over the same time period. Male sex, neutering, delayed physeal closure and breed-specific high body weight may play an important role in the pathogenesis of SCFE in Maine Coon cats.

Evaluation of Presumably Disease Causing SCN1A Variants in a Cohort of Common Epilepsy Syndromes.

OBJECTIVE: The SCN1A gene, coding for the voltage-gated Na+ channel alpha subunit NaV1.1, is the clinically most relevant epilepsy gene. With the advent of high-throughput next-generation sequencing, clinical laboratories are generating an ever-increasing catalogue of SCN1A variants. Variants are more likely to be classified as pathogenic if they have already been identified previously in a patient with epilepsy. Here, we critically re-evaluate the pathogenicity of this class of variants in a cohort of patients with common epilepsy syndromes and subsequently ask whether a significant fraction of benign variants have been misclassified as pathogenic. METHODS: We screened a discovery cohort of 448 patients with a broad range of common genetic epilepsies and 734 controls for previously reported SCN1A mutations that were assumed to be disease causing. We re-evaluated the evidence for pathogenicity of the identified variants using in silico predictions, segregation, original reports, available functional data and assessment of allele frequencies in healthy individuals as well as in a follow up cohort of 777 patients. RESULTS AND INTERPRETATION: We identified 8 known missense mutations, previously reported as pathogenic, in a total of 17 unrelated epilepsy patients (17/448; 3.80%). Our re-evaluation indicates that 7 out of these 8 variants (p.R27T; p.R28C; p.R542Q; p.R604H; p.T1250M; p.E1308D; p.R1928G; NP_001159435.1) are not pathogenic. Only the p.T1174S mutation may be considered as a genetic risk factor for epilepsy of small effect size based on the enrichment in patients (P = 6.60 x 10-4; OR = 0.32, fishers exact test), previous functional studies but incomplete penetrance. Thus, incorporation of previous studies in genetic counseling of SCN1A sequencing results is challenging and may produce incorrect conclusions.

Efficacy and safety of cyclic pyranopterin monophosphate substitution in severe molybdenum cofactor deficiency type A: a prospective cohort study.

BACKGROUND: Molybdenum cofactor deficiency (MoCD) is characterised by early, rapidly progressive postnatal encephalopathy and intractable seizures, leading to severe disability and early death. Previous treatment attempts have been unsuccessful. After a pioneering single treatment we now report the outcome of the complete first cohort of patients receiving substitution treatment with cyclic pyranopterin monophosphate (cPMP), a biosynthetic precursor of the cofactor. METHODS: In this observational prospective cohort study, newborn babies with clinical and biochemical evidence of MoCD were admitted to a compassionate-use programme at the request of their treating physicians. Intravenous cPMP (80-320 µg/kg per day) was started in neonates diagnosed with MoCD (type A and type B) following a standardised protocol. We prospectively monitored safety and efficacy in all patients exposed to cPMP. FINDINGS: Between June 6, 2008, and Jan 9, 2013, intravenous cPMP was started in 16 neonates diagnosed with MoCD (11 type A and five type B) and continued in eight type A patients for up to 5 years. We observed no drug-related serious adverse events after more than 6000 doses. The disease biomarkers urinary S-sulphocysteine, xanthine, and urate returned to almost normal concentrations in all type A patients within 2 days, and remained normal for up to 5 years on continued cPMP substitution. Eight patients with type A disease rapidly improved under treatment and convulsions were either completely suppressed or substantially reduced. Three patients treated early remain seizure free and show near-normal long-term development. We detected no biochemical or clinical response in patients with type B disease. INTERPRETATION: cPMP substitution is the first effective therapy for patients with MoCD type A and has a favourable safety profile. Restoration of molybdenum cofactor-dependent enzyme activities results in a greatly improved neurodevelopmental outcome when started sufficiently early. The possibility of MoCD type A needs to be urgently explored in every encephalopathic neonate to avoid any delay in appropriate cPMP substitution, and to maximise treatment benefit. FUNDING: German Ministry of Education and Research; Orphatec/Colbourne Pharmaceuticals.

Identification of a new genomic hot spot of evolutionary diversification of protein function.

Establishment of phylogenetic relationships remains a challenging task because it is based on computational analysis of genomic hot spots that display species-specific sequence variations. Here, we identify a species-specific thymine-to-guanine sequence variation in the Glrb gene which gives rise to species-specific splice donor sites in the Glrb genes of mouse and bushbaby. The resulting splice insert in the receptor for the inhibitory neurotransmitter glycine (GlyR) conveys synaptic receptor clustering and specific association with a particular synaptic plasticity-related splice variant of the postsynaptic scaffold protein gephyrin. This study identifies a new genomic hot spot which contributes to phylogenetic diversification of protein function and advances our understanding of phylogenetic relationships.

Rare variants in γ-aminobutyric acid type A receptor genes in rolandic epilepsy and related syndromes.

OBJECTIVE: To test whether mutations in γ-aminobutyric acid type A receptor (GABAA -R) subunit genes contribute to the etiology of rolandic epilepsy (RE) or its atypical variants (ARE). METHODS: We performed exome sequencing to compare the frequency of variants in 18 GABAA -R genes in 204 European patients with RE/ARE versus 728 platform-matched controls. Identified GABRG2 variants were functionally assessed for protein stability, trafficking, postsynaptic clustering, and receptor function. RESULTS: Of 18 screened GABAA -R genes, we detected an enrichment of rare variants in the GABRG2 gene in RE/ARE patients (5 of 204, 2.45%) in comparison to controls (1 of 723, 0.14%; odds ratio = 18.07, 95% confidence interval = 2.01-855.07, p = 0.0024, pcorr = 0.043). We identified a GABRG2 splice variant (c.549-3T>G) in 2 unrelated patients as well as 3 nonsynonymous variations in this gene (p.G257R, p.R323Q, p.I389V). Functional assessment showed reduced surface expression of p.G257R and decreased GABA-evoked currents for p.R323Q. The p.G257R mutation displayed diminished levels of palmitoylation, a post-translational modification crucial for trafficking of proteins to the cell membrane. Enzymatically raised palmitoylation levels restored the surface expression of the p.G257R variant γ2 subunit. INTERPRETATION: The statistical association and the functional evidence suggest that mutations of the GABRG2 gene may increase the risk of RE/ARE. Restoring the impaired membrane trafficking of some GABRG2 mutations by enhancing palmitoylation might be an interesting therapeutic approach to reverse the pathogenic effect of such mutants.

A sensitive and stable amperometric nitrate biosensor employing Arabidopsis thaliana nitrate reductase.

Nitrate reductase (NR) from the plant Arabidopsis thaliana has been employed in the development of an amperometric nitrate biosensor that functions at physiological pH. The anion anthraquinone-2-sulfonate (AQ) is used as an effective artificial electron transfer partner for NR at a glassy carbon (GC) electrode. Nitrate is enzymatically reduced to nitrite and the oxidized form of NR is electrochemically reduced by the hydroquinone form of the mediator (AQH2). The GC/NR electrode shows a pronounced cathodic wave for nitrate reduction and the catalytic current increases linearly in the nitrate concentration range of 10-400 µM with a correlation coefficient of 0.989. Using an amperometric method, a low detection limit of 0.76 nM (S/N = 3) was achieved. The practical application of the present electrochemical biosensor was demonstrated by the determination of nitrate concentration in natural water samples and the results agreed well with a standard spectroscopic method.

Exonic microdeletions of the gephyrin gene impair GABAergic synaptic inhibition in patients with idiopathic generalized epilepsy.

Gephyrin is a postsynaptic scaffolding protein, essential for the clustering of glycine and γ-aminobutyric acid type-A receptors (GABAARs) at inhibitory synapses. An impairment of GABAergic synaptic inhibition represents a key pathway of epileptogenesis. Recently, exonic microdeletions in the gephyrin (GPHN) gene have been associated with neurodevelopmental disorders including autism spectrum disorder, schizophrenia and epileptic seizures. Here we report the identification of novel exonic GPHN microdeletions in two patients with idiopathic generalized epilepsy (IGE), representing the most common group of genetically determined epilepsies. The identified GPHN microdeletions involve exons 5-9 (Δ5-9) and 2-3 (Δ2-3), both affecting the gephyrin G-domain. Molecular characterization of the GPHN Δ5-9 variant demonstrated that it perturbs the clustering of regular gephyrin at inhibitory synapses in cultured mouse hippocampal neurons in a dominant-negative manner, resulting in a significant loss of γ2-subunit containing GABAARs. GPHN Δ2-3 causes a frameshift resulting in a premature stop codon (p.V22Gfs*7) leading to haplo-insufficiency of the gene. Our results demonstrate that structural exonic microdeletions affecting the GPHN gene constitute a rare genetic risk factor for IGE and other neuropsychiatric disorders by an impairment of the GABAergic inhibitory synaptic transmission.

Pulsed electron paramagnetic resonance spectroscopy of (33)S-labeled molybdenum cofactor in catalytically active bioengineered sulfite oxidase.

Molybdenum enzymes contain at least one pyranopterin dithiolate (molybdopterin, MPT) moiety that coordinates Mo through two dithiolate (dithiolene) sulfur atoms. For sulfite oxidase (SO), hyperfine interactions (hfi) and nuclear quadrupole interactions (nqi) of magnetic nuclei (I ≠ 0) near the Mo(V) (d(1)) center have been measured using high-resolution pulsed electron paramagnetic resonance (EPR) methods and interpreted with the help of density functional theory (DFT) calculations. These have provided important insights about the active site structure and the reaction mechanism of the enzyme. However, it has not been possible to use EPR to probe the dithiolene sulfurs directly since naturally abundant (32)S has no nuclear spin (I = 0). Here we describe direct incorporation of (33)S (I = 3/2), the only stable magnetic sulfur isotope, into MPT using controlled in vitro synthesis with purified proteins. The electron spin echo envelope modulation (ESEEM) spectra from (33)S-labeled MPT in this catalytically active SO variant are dominated by the "interdoublet" transition arising from the strong nuclear quadrupole interaction, as also occurs for the (33)S-labeled exchangeable equatorial sulfite ligand [ Klein, E. L., et al. Inorg. Chem. 2012 , 51 , 1408 - 1418 ]. The estimated experimental hfi and nqi parameters for (33)S (aiso = 3 MHz and e(2)Qq/h = 25 MHz) are in good agreement with those predicted by DFT. In addition, the DFT calculations show that the two (33)S atoms are indistinguishable by EPR and reveal a strong intermixing between their out-of-plane pz orbitals and the dxy orbital of Mo(V).

Changes in neural network homeostasis trigger neuropsychiatric symptoms.

The mechanisms that regulate the strength of synaptic transmission and intrinsic neuronal excitability are well characterized; however, the mechanisms that promote disease-causing neural network dysfunction are poorly defined. We generated mice with targeted neuron type-specific expression of a gain-of-function variant of the neurotransmitter receptor for glycine (GlyR) that is found in hippocampectomies from patients with temporal lobe epilepsy. In this mouse model, targeted expression of gain-of-function GlyR in terminals of glutamatergic cells or in parvalbumin-positive interneurons persistently altered neural network excitability. The increased network excitability associated with gain-of-function GlyR expression in glutamatergic neurons resulted in recurrent epileptiform discharge, which provoked cognitive dysfunction and memory deficits without affecting bidirectional synaptic plasticity. In contrast, decreased network excitability due to gain-of-function GlyR expression in parvalbumin-positive interneurons resulted in an anxiety phenotype, but did not affect cognitive performance or discriminative associative memory. Our animal model unveils neuron type-specific effects on cognition, formation of discriminative associative memory, and emotional behavior in vivo. Furthermore, our data identify a presynaptic disease-causing molecular mechanism that impairs homeostatic regulation of neural network excitability and triggers neuropsychiatric symptoms.

Multivalent interactions of the SUMO-interaction motifs in RING finger protein 4 determine the specificity for chains of the SUMO.

RNF4 (RING finger protein 4) is a STUbL [SUMO (small ubiquitin-related modifier)-targeted ubiquitin ligase] controlling PML (promyelocytic leukaemia) nuclear bodies, DNA double strand break repair and other nuclear functions. In the present paper, we describe that the sequence and spacing of the SIMs (SUMO-interaction motifs) in RNF4 regulate the avidity-driven recognition of substrate proteins carrying SUMO chains of variable length.

Mediated electrochemistry of nitrate reductase from Arabidopsis thaliana.

Herein we report the mediated electrocatalytic voltammetry of the plant molybdoenzyme nitrate reductase (NR) from Arabidopsis thaliana using the established truncated molybdenum-heme fragment at a glassy carbon (GC) electrode. Methyl viologen (MV), benzyl viologen (BV), and anthraquinone-2-sulfonic acid (AQ) are employed as effective artificial electron transfer partners for NR, differing in redox potential over a range of about 220 mV and delivering different reductive driving forces to the enzyme. Nitrate is reduced at the Mo active site of NR, yielding the oxidized form of the enzyme, which is reactivated by the electro-reduced form of the mediator. Digital simulation was performed using a single set of enzyme dependent parameters for all catalytic voltammetry obtained under different sweep rates and various substrate or mediator concentrations. The kinetic constants from digital simulation provide new insight into the kinetics of the NR catalytic mechanism.