A perspective on In vitro developmental neurotoxicity test assay results: An expert panel review.

For decades, there has been increasing concern about the potential developmental neurotoxicity (DNT) associated with chemicals. Regulatory agencies have historically utilized standardized in vivo testing to evaluate DNT. Owing to considerations including higher-throughput screening for DNT, reduction in animal use, and potential cost efficiencies, the development of alternative new approach methods (NAMs) occurred; specifically, the advent of the DNT in vitro test battery (DNT IVB). SciPinion convened an expert panel to address specific questions related to the interpretation of in vitro DNT test data. The consensus of the expert panel was that the DNT IVB might be used during initial screening, but it is not presently a complete or surrogate approach to determine whether a chemical is a DNT in humans. By itself, the DNT IVB does not have the ability to capture nuances and complexity of the developing nervous system and associated outcomes including behavioral ontogeny, motor activity, sensory function, and learning/memory. Presently, such developmental landmarks cannot be adequately assessed in the DNT IVB or by other NAMs. The expert panel (all who serve as co-authors of this review) recommended that additional data generation and validation is required before the DNT IVB can be considered for application within global regulatory frameworks for decision-making.

Grouping of PFAS for human health risk assessment: Findings from an independent panel of experts.

An expert panel was convened to provide insight and guidance on per- and polyfluoroalkyl substances (PFAS) grouping for the purposes of protecting human health from drinking water exposures, and how risks to PFAS mixtures should be assessed. These questions were addressed through multiple rounds of blind, independent responses to charge questions, and review and comments on co-panelists responses. The experts agreed that the lack of consistent interpretations of human health risk for well-studied PFAS and the lack of information for the vast majority of PFAS present significant challenges for any mixtures risk assessment approach. Most experts agreed that "all PFAS" should not be grouped together, persistence alone is not sufficient for grouping PFAS for the purposes of assessing human health risk, and that the definition of appropriate subgroups can only be defined on a case-by-case manner. Most panelists agreed that it is inappropriate to assume equal toxicity/potency across the diverse class of PFAS. A tiered approach combining multiple lines of evidence was presented as a possible viable means for addressing PFAS that lack analytical and/or toxicological studies. Most PFAS risk assessments will need to employ assumptions that are more likely to overestimate risk than to underestimate risk, given the choice of assumptions regarding dose-response model, uncertainty factors, and exposure information.

Glucose challenge test screening for prediabetes and early diabetes.

AIMS: To test the hypothesis that a 50-g oral glucose challenge test with 1-h glucose measurement would have superior performance compared with other opportunistic screening methods. METHODS: In this prospective study in a Veterans Health Administration primary care clinic, the following test performances, measured by area under receiver-operating characteristic curves, were compared: 50-g oral glucose challenge test; random glucose; and HbA1c level, using a 75-g oral glucose tolerance test as the 'gold standard'. RESULTS: The study population was comprised of 1535 people (mean age 56 years, BMI 30.3 kg/m2 , 94% men, 74% black). By oral glucose tolerance test criteria, diabetes was present in 10% and high-risk prediabetes was present in 22% of participants. The plasma glucose challenge test provided area under receiver-operating characteristic curves of 0.85 (95% CI 0.78-0.91) to detect diabetes and 0.76 (95% CI 0.72-0.80) to detect high-risk dysglycaemia (diabetes or high-risk prediabetes), while area under receiver-operating characteristic curves for the capillary glucose challenge test were 0.82 (95% CI 0.75-0.89) and 0.73 (95% CI 0.69-0.77) for diabetes and high-risk dysglycaemia, respectively. Random glucose performed less well [plasma: 0.76 (95% CI 0.69-0.82) and 0.66 (95% CI 0.62-0.71), respectively; capillary: 0.72 (95% CI 0.65-0.80) and 0.64 (95% CI 0.59-0.68), respectively], and HbA1c performed even less well [0.67 (95% CI 0.57-0.76) and 0.63 (95% CI 0.58-0.68), respectively]. The cost of identifying one case of high-risk dysglycaemia with a plasma glucose challenge test would be $42 from a Veterans Health Administration perspective, and $55 from a US Medicare perspective. CONCLUSIONS: Glucose challenge test screening, followed, if abnormal, by an oral glucose tolerance test, would be convenient and more accurate than other opportunistic tests. Use of glucose challenge test screening could improve management by permitting earlier therapy.

Survival outcomes in patients with early stage, resected pancreatic cancer - a comparison of gemcitabine- and 5-fluorouracil-based chemotherapy and chemoradiation regimens.

PURPOSE: We conducted a comparative survival analysis between patients with resected pancreatic cancer who received adjuvant treatment with either gemcitabine- or 5-fluorouracil-based chemotherapy and chemoradiation regimens. PATIENTS AND METHODS: The Surveillance, Epidemiology and End Results (SEER)-Medicare database was used to identify patients with pancreatic cancer diagnosed from 1998 to 2005 who received curative surgery and adjuvant chemotherapy with either 5-fluorouracil or gemcitabine. These groups were subdivided by treatment with radiotherapy. Patients were followed until death, study end-point or a maximum of 5 years after diagnosis. RESULTS: Three hundred and fifty-nine patients received 5-fluorouracil and 346 received gemcitabine. Compared with chemoradiation with 5-fluorouracil, outcomes for patients who received chemoradiation with gemcitabine did not differ. Patients who received gemcitabine without radiation had increased hazards (poorly differentiated tumours: HR = 1.50, p = 0.01; moderately differentiated tumours, HR = 1.28, p = 0.11). However, outcomes of patients who received 5-fluorouracil without radiation varied with tumour grade. In moderately differentiated tumours, patients had better outcomes with 5-fluorouracil when compared with chemoradiation with 5-fluorouracil (HR = 0.42, p = 0.02). In poorly differentiated tumours, the opposite was true (HR 2.10, p = 0.09). CONCLUSION: Patients with low-grade resected pancreatic cancer may have better outcomes with 5-fluorouracil-based chemotherapy without radiation when compared with 5-fluorouracil with radiation.

Occupational blood exposure among unlicensed home care workers and home care registered nurses: are they protected?

BACKGROUND: Little is known about the risk of blood exposure among personnel providing care to individual patients residing at home. The objective of this study was to document and compare blood exposure risks among unlicensed home care personal care assistants (PCAs) and home care registered nurses (RNs). METHODS: PCAs self-completed surveys regarding blood and body fluid (BBF) contact in group settings (n = 980), while RNs completed mailed surveys (n = 794). RESULTS: PCAs experience BBF contact in the course of providing care for home-based clients at a rate approximately 1/3 the rate experienced by RNs providing home care (8.1 and 26.7 per 100 full time equivalent (FTE), respectively), and the majority of PCA contact episodes did not involve direct sharps handling. However, for PCAs who performed work activities such as handling sharps and changing wound dressings, activities much more frequently performed by RNs, PCAs were at increased risk of injury when compared with RNs (OR = 7.4 vs. 1.4) and (OR = 6.3 vs. 2.5), respectively. CONCLUSION: Both PCAs and RNs reported exposures to sharps, blood, and body fluids in the home setting at rates that warrant additional training, prevention, and protection. PCAs appear to be at increased risk of injury when performing nursing-related activities for which they are inexperienced and/or lack training. Further efforts are needed to protect home care workers from blood exposure, namely by assuring coverage and enforcement of the Occupational Safety and Health Administration (OSHA) Bloodborne Pathogen Standard [Occupational Safety and Health Administration. 1993. Frequently Asked Questions Concerning the Bloodborne Pathogens Standard. Available at: http://www.osha.gov/pls/oshaweb/owadisp.show_document?p_table=INTERPRETATIONS &p_id=21010#Scope. Accessed May 30, 2008].

Covariation of human microsomal protein per gram of liver with age: absence of influence of operator and sample storage may justify interlaboratory data pooling.

Scaling of metabolic clearance values from liver microsomal data or recombinantly expressed cytochrome P450 enzymes to predict human hepatic clearance requires knowledge of the amount of microsomal protein per gram of liver (MPPGL). Identification of physiological covariates of MPPGL requires analysis of values from large diverse populations, which necessitates pooling of data from numerous sources. To ensure compatibility between results obtained within and between studies, the impact of interoperator differences and sample storage on values of MPPGL was investigated. With use of triplicate samples from one liver (HL86), no statistically significant difference was detected between values of MPPGL prepared from samples stored at -80 degrees C (23.5 +/- 1.2 mg g(-1)) and those determined using fresh tissue (21.9 +/- 0.3 mg g(-1)). Although there was a significant difference in the yield of microsomal protein obtained from another liver sample (HL43) by three different operators (17 +/- 1, 19 +/- 2, and 24 +/- 1 mg g(-1); p = 0.004, analysis of variance), no difference was observed in the estimated MPPGL after application of appropriate correction factors for each operator (28 +/- 1, 30 +/- 5, and 31 +/- 4 mg g(-1)). The result provided justification for pooling reported values of MPPGL for use in covariate analysis. Investigation of the relationship between age and MPPGL provided preliminary evidence that MPPGL values increase from birth to a maximum of 40 mg g(-1) [95% confidence interval for the geometric mean (95% CI mean(geo)): 37-43 mg g(-1) at approximately 28 years followed by a gradual decrease in older age (mean of 29 mg g(-1) at 65 years; 95% CI mean(geo): 27-32 mg g(-1)). Accordingly, appropriate age-adjusted scaling factors should be used in extrapolating in vitro clearance values to clinical studies.

Finding intermediates in the O2 activation pathways of non-heme iron oxygenases.

Intermediates in the reaction cycle of an oxygenase are usually very informative with respect to the chemical mechanism of O 2 activation and insertion. However, detection of these intermediates is often complicated by their short lifetime and the regulatory mechanism of the enzyme designed to ensure specificity. Here, the methods used to detect the intermediates in an extradiol dioxygenase, a Rieske cis-dihydrodiol dioxygenase, and soluble methane monooxygenase are discussed. The methods include the use of alternative, chromophoric substrates, mutagenesis of active site catalytic residues, forced changes in substrate binding order, control of reaction rates using regulatory proteins, and initialization of catalysis in crystallo.

Molecular architecture and conformational flexibility of human RNA polymerase II.

Transcription by RNA polymerase II (RNAPII) is a central process in eukaryotic gene regulation. While atomic details exist for the yeast RNAPII, characterization of the human complex lags behind, mostly due to the inability to obtain large quantities of purified material. Although the complexes have the same protein composition and high sequence similarity, understanding of transcription and of transcription-coupled DNA repair (TCR) in humans will require the use of human proteins in structural studies. We have used cryo-electron microscopy, image reconstruction, and variance analysis to characterize the structure and dynamics of human RNAPII (hRNAPII). Our studies show that hRNAPII in solution parallels the conformational flexibility of the yeast structures crystallized in different states but also illustrate a more extensive conformational range with potential biological significance. This hRNAPII study will serve as a structural platform to build up higher-order transcription and TCR complexes and to gain information that may be unique to the human RNAPII system.

Application of QSTRs in the selection of a surrogate toxicity value for a chemical of concern.

As part of the EPA's mission to protect the environment, chemicals of concern (CoCs) at Superfund or other hazardous waste sites are cleaned up based on their potential toxicity to humans and the surrounding ecosystem. Oftentimes, there is a lack of experimental toxicity data to assess the health effects for a CoC in the literature. This research describes a method using Quantitative Structure Toxicity Relationships (QSTRs) for identifying a surrogate chemical for any given CoC. The toxicity data of the surrogate chemical can then be used to rank hazardous waste-site chemicals prior to cleanup decisions. A commercial QSTR model, TOPKAT, was used to establish structural and descriptor similarity between the CoC and the compounds in the QSTR model database using the Oral Rat Chronic LOAEL model. All database chemicals within a similarity distance of < or = 0.200 from the CoC are considered as potential surrogates. If the CoC fails to satisfy model considerations for the LOAEL model, no surrogate is suggested. Potential surrogates that have toxicity data on Integrated Risk Information System (IRIS), Health Effects Assessment Summary Tables (HEAST), or National Center for Environmental Assessment (NCEA) provisional toxicity value list become candidate surrogates. If more than one candidate surrogate is identified, the chemical with the most conservative RfD is suggested as the surrogate. The procedure was applied to determine an appropriate surrogate for dichlorobenzophenone (DCBP), a metabolite of chlorobenzilate, dichlorodiphenyltrichloroethane, and dicofol. Forty-seven potential surrogates were identified that were within the similarity distance of < or = 0.200, of which only five chemicals had an RfD on IRIS, HEAST, or on the NCEA provisional toxicity value list. Among the five potential surrogates, chlorobenzilate with an RfD of 2 x 10(-2) mg/kg-day was chosen as a surrogate for DCBP as it had the most conservative toxicity value. This compared well with surrogate selection using available metabolic information for DCBP and its metabolites or parent compounds in the literature and the provisional toxicity value of 3 x 10(-2) mg/kg-day that NCEA developed using a subchronic study.

Racial differences in survival post cerebral infarction among the elderly.

OBJECTIVE: To investigate whether there are differences in poststroke survival between African American and white patients, aged 65 and over, in the United States. METHODS: A biracial cohort of patients was selected from a random 20% national sample of Medicare patients (age 65 and over) hospitalized with cerebral infarction in 1991, and was followed up to a period of 3 years. The Cox regression model was used for covariate adjustment. RESULTS: A total of 47,045 patients (including 5,324 African Americans) were identified for our analysis. Compared to white patients, African American patients on average were 6% more likely to die post cerebral infarction. The subpopulation analyses further suggest that African Americans age 65 to 74 had much lower 3-year survival probabilities (15 to 20%) than their white counterparts. CONCLUSIONS: The authors find evidence of racial disparities in survival post cerebral infarction among the elderly, although the differences by race are not as great as reported elsewhere for stroke incidence and mortality. Future analyses, using more clinically detailed data, should focus especially on whether survival differences by race persist in the young-old (age 65 to 74) population.

Intermediate Q from soluble methane monooxygenase hydroxylates the mechanistic substrate probe norcarane: evidence for a stepwise reaction.

Norcarane is a valuable mechanistic probe for enzyme-catalyzed hydrocarbon oxidation reactions because different products or product distributions result from concerted, radical, and cation based reactions. Soluble methane monooxygenase (sMMO) from Methylosinus trichosporium OB3b catalyzes the oxidation of norcarane to afford 3-hydroxymethylcyclohexene and 3-cycloheptenol, compounds characteristic of radical and cationic intermediates, respectively, in addition to 2- and 3-norcaranols. Past single turnover transient kinetic studies have identified several optically distinct intermediates from the catalytic cycle of the hydroxylase component of sMMO. Thus, the reaction between norcarane and key reaction intermediates can be directly monitored. The presence of norcarane increases the rate of decay of only one intermediate, the high-valent bis-mu-oxo Fe(IV)(2) cluster-containing species compound Q, showing that it is responsible for the majority of the oxidation chemistry. The observation of products from both radical and cationic intermediates from norcarane oxidation catalyzed by sMMO is consistent with a mechanism in which an initial substrate radical intermediate is formed by hydrogen atom abstraction. This intermediate then undergoes either oxygen rebound, intramolecular rearrangement followed by oxygen rebound, or loss of a second electron to yield a cationic intermediate to which OH(-) is transferred. The estimated lower limit of 20 ps for the lifetime of the putative radical intermediate is in accord with values determined from previous studies of sterically hindered sMMO probes.

Malignant teratoma of the thyroid with primitive neuroepithelial and mesenchymal sarcomatous components.

A 15-year-old black girl had a near total resection of a malignant thyroid teratoma with bilateral nodal involvement and mediastinal extension. A predominant neuroepithelial pattern had ependymal rosettes and mitoses, stained for neuron-specific enolase, neuron-specific B tubulin, and synaptophysin. A malignant spindle cell component stained for smooth-muscle actin, muscle actin, and to a lesser extent S-100. Loose myxoid tissue resembled primitive cartilage. Epithelial membrane antigen and cytokeratin identified epithelial foci. Chromogranin A, MIC2, glial fibrillary acidic protein, and thyroid stimulating hormone receptor stains were negative. There was focal anaplasia. DNA ploidy by laser scanning cytometry was 1.2. The tumor from the left and right thyroid lobes exhibited trisomy 8, the right also had hyperdiploid cell lines. She was treated with aggressive combination chemotherapy and radiation. Presently there is no residual disease 16 months after diagnosis. Malignant thyroid teratoma is an aggressive tumor, with 15 of 27 reported patients dying 2 weeks to 3 years after diagnosis. Survivors have been treated with total or subtotal resection, combination chemotherapy with agents effective in the treatment of germ cell tumors as well as sarcomas, and radiation for either recurrent or residual disease. The heterologous elements, lacking MIC2 staining and t(11;22), support the diagnosis of malignant teratoma rather than a neuroepithelial tumor. Trisomy 8 is the first cytogenetic abnormality described in malignant thyroid teratoma. Therapy should be tailored to the management of all transformed histologies.

Desaturation reactions catalyzed by soluble methane monooxygenase.

Soluble methane monooxygenase (MMO) is shown to be capable of catalyzing desaturation reactions in addition to the usual hydroxylation and epoxidation reactions. Dehydrogenated products are generated from MMO-catalyzed oxidation of certain substrates including ethylbenzene and cyclohexadienes. In the reaction of ethylbenzene, desaturation of ethyl C-H occurred along with the conventional hydroxvlations of ethyl and phenyl C-Hs. As a result, styrene is formed together with ethylphenols and phenylethanols. Similarly, when 1,3- and 1,4-cyclohexadienes were used as substrates, benzene was detected as a product in addition to the corresponding alcohols and epoxides. In all cases, reaction conditions were found to significantly affect the distribution among the different products. This new activity of MMO is postulated to be associated with the chemical properties of the substrates rather than fundamental changes in the nature of the oxygen and C-H activation chemistries. The formation of the desaturated products is rationalized by formation of a substrate cationic intermediate, possibly via a radical precursor. The cationic species is then proposed to partition between recombination (alcohol formation) and elimination (alkene production) pathways. This novel function of MMO indicates close mechanistic kinship between the hydroxylation and desaturation reactions catalyzed by the nonheme diiron clusters.

Residues in Methylosinus trichosporium OB3b methane monooxygenase component B involved in molecular interactions with reduced- and oxidized-hydroxylase component: a role for the N-terminus.

Methane monooxygenase (MMO) is a non-heme-iron-containing enzyme which consists of 3 protein components: a hydroxylase (MMOH), an NAD(P)H-linked reductase (MMOR), and a 138-residue regulatory protein, component B (MMOB). Here, NMR spectroscopy has been used to derive interactions between MMOB and reduced and oxidized states of MMOH (245 kDa). Differential broadening of MMOB resonances in 1H-15N HSQC spectra acquired at different molar ratios of MMOH indicates interaction of both proteins, with MMOB binding more tightly to oxidized MMOH as observed previously. The most broadened backbone NH resonances suggest which residues in MMOB are part of the MMOH-binding interface, particularly when those residues are spatially close or clustered in the structure of MMOB. Although a number of different residues in MMOB appear to be involved in interacting with oxidized- and reduced-MMOH, some are identical. The two most common segments, proximal in the structure of MMOB, are beta-strand 1 with turn 1 (residues 36-46) and alpha-helix 3 going into loop 2 (residues 101-112). In addition, the N-terminus of MMOB is observed to be involved in binding to MMOH in either redox state. This is most strongly evidenced by use of a synthetic N-terminal peptide from MMOB (residues 1-29) in differential broadening 1H TOCSY studies with MMOH. Binding specificity is demonstrated by displacement of the peptide from MMOH by parent MMOB, indicating that the peptide binds in or near the normal site of N-terminal binding. The N-terminus is also observed to be functionally important. Steady-state kinetic studies show that neither a delta2-29 MMOB deletion mutant (which in fact does bind to MMOH), the N-terminal peptide, nor a combination of the two elicit the effector functions of MMOB. Furthermore, transient kinetic studies indicate that none of the intermediates of the MMOH catalytic cycle are observed if either the delta2-29 MMOB mutant or the N-terminal peptide is used in place of MMOB, suggesting that deletion of the N-terminus prevents reaction of reduced MMOH with O2 that initiates catalysis.

Methane monooxygenase component B mutants alter the kinetics of steps throughout the catalytic cycle.

Component interactions play important roles in the regulation of catalysis by methane monooxygenase (MMO). The binding of component B (MMOB) to the hydroxylase component (MMOH) has been shown in previous studies to cause structural changes in MMOH that result in altered thermodynamic and kinetic properties during the reduction and oxygen binding steps of the catalytic cycle. Here, specific amino acid residues of MMOB that play important roles in the interconversion of several intermediates of the MMO cycle have been identified. Both of the histidine residues in Methylosinus trichosporium OB3b MMOB (H5 and H33) were chemically modified by diethylpyrocarbonate (DEPC). Although the DEPC--MMOB species exhibited only minor changes relative to unmodified MMOB in steady-state MMO turnover, large decreases in the formation rate constants of the reaction cycle intermediates, compound P and compound Q, were observed. The site specific mutants H5A, H33A, and H5A/H33A were made and characterized. H5A and wild type MMOB elicited similar steady-state and transient kinetics, although the mutant caused a slightly lower rate constant for Q formation. Conversely, H33A exhibited a >50-fold decrease in the P formation rate constant, which resulted in slower formation of Q. The kinetics of the double mutant (H5A/H33A) were similar to those of H33A, suggesting that the highly conserved residue, H33, has the most significant effect on the efficient progress of the cycle. Ongoing NMR investigations of residues perturbed by formation of the MMOH-MMOB complex suggested construction of the MMOB N107G/S109A/S110A/T111A quadruple mutant. This mutant was found to elicit a nearly 2-fold increase in specific activity for steady-state MMO turnover of large substrates such as furan and nitrobenzene but caused no similar increase for the physiological substrate, methane. While the quadruple mutant did not have a significant effect on P and Q formation, it caused an almost 3-fold increase in the decay rate constant of Q for furan oxidation and a 2-fold faster product release rate constant for p-nitrophenol resulting from nitrobenzene oxidation. Conversely, this mutant caused the Q decay rate constant to decrease 7-fold for methane oxidation but left the product release step unaffected. These results show for the first time that MMOB exerts influence at late as well as early steps in the catalytic cycle. They also suggest that MMOB plays a critical role in determining the ability of MMO to distinguish between methane and larger substrates.

Physically demanding work and inadequate sleep, pain medication use, and absenteeism in registered nurses.

Pain and fatigue are early indicators of musculoskeletal strain. This study examined associations among eight physical demands and inadequate sleep, pain medication use, and absenteeism in 3727 working registered nurses (RNs). Among the demands, awkward head/arm postures were associated with each outcome (inadequate sleep: odds ratio [OR], 1.96; 95% confidence interval [CI], 1.41 to 2.72; pain medication: OR, 1.65; CI, 1.12 to 2.24; absenteeism: OR, 1.60; CI, 1.26 to 2.04). A dose-response relationship was present; as the number of demands increased, the likelihood of each outcome increased. Odds ratios for eight demands versus no demands were as follows: inadequate sleep (OR, 5.88; CI, 2.30 to 15.50), pain medication (OR, 3.30; CI, 1.34 to 8.11), and absenteeism (OR, 2.13; CI, 1.15 to 3.94). Adjustment using multiple logistic regression for lifestyle, demographics, and work schedule did little to alter the findings. Interventions to promote nurses' health should limit the physical demands of the work.

The burden of illness of cancer: economic cost and quality of life.

Cancer is a major public health issue and represents a significant burden of disease. In this chapter, we analyze the main measures of burden of disease as relate to cancer. Specifically, we review incidence and mortality, years of life lost from cancer, and cancer prevalence. We also discuss the economic burden of cancer, including cost of illness, phase-specific and long-term costs, and indirect costs. We then examine the impact of cancer on health-related quality of life as measured in global terms (disability-adjusted life years and quality-adjusted life years) and using evaluation-oriented applications of health-related quality of life scales. Throughout, we note the relative strengths and weaknesses of the various approaches to measuring the burden of cancer as well as the methodologic challenges that persist in burden-of-illness research. We conclude with a discussion of the research agenda to improve our understanding of the burden of cancer and of illness more generally.

Single turnover chemistry and regulation of O2 activation by the oxygenase component of naphthalene 1,2-dioxygenase.

Naphthalene 1,2-dioxygenase (NDOS) is a three-component enzyme that catalyzes cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene formation from naphthalene, O2, and NADH. We have determined the conditions for a single turnover of NDOS for the first time and studied the regulation of catalysis. As isolated, the alpha3beta3 oxygenase component (NDO) has up to three catalytic pairs of metal centers (one mononuclear Fe2+ and one diferric Rieske iron-sulfur cluster). This form of NDO is unreactive with O2. However, upon reduction of the Rieske cluster and exposure to naphthalene and O2, approximately 0.85 cis-diol product per occupied mononuclear iron site rapidly forms. Substrate binding is required for oxygen reactivity. Stopped-flow and chemical quench analyses indicate that the rate constant of the single turnover product-forming reaction significantly exceeds the NDOS turnover number. UV-visible and electron paramagnetic resonance spectroscopies show that during catalysis, one mononuclear iron and one Rieske cluster are oxidized per product formed, satisfying the two-electron reaction stoichiometry. The addition of oxidized or reduced NDOS ferredoxin component (NDF) increases both the product yield and rate of oxidation of formerly unreactive Rieske clusters. The results show that NDO alone catalyzes dioxygenase chemistry, whereas NDF appears to serve only an electron transport role, in this case redistributing electrons to competent active sites.