Contrasting Mechanisms of Aromatic and Aryl-Methyl Substituent Hydroxylation by the Rieske Monooxygenase Salicylate 5-Hydroxylase.

The hydroxylase component (S5HH) of salicylate-5-hydroxylase catalyzes C5 ring hydroxylation of salicylate but switches to methyl hydroxylation when a C5 methyl substituent is present. The use of 18O2 reveals that both aromatic and aryl-methyl hydroxylations result from monooxygenase chemistry. The functional unit of S5HH comprises a nonheme Fe(II) site located 12 Šacross a subunit boundary from a one-electron reduced Rieske-type iron-sulfur cluster. Past studies determined that substrates bind near the Fe(II), followed by O2 binding to the iron to initiate catalysis. Stopped-flow-single-turnover reactions (STOs) demonstrated that the Rieske cluster transfers an electron to the iron site during catalysis. It is shown here that fluorine ring substituents decrease the rate constant for Rieske electron transfer, implying a prior reaction of an Fe(III)-superoxo intermediate with a substrate. We propose that the iron becomes fully oxidized in the resulting Fe(III)-peroxo-substrate-radical intermediate, allowing Rieske electron transfer to occur. STO using 5-CD3-salicylate-d8 occurs with an inverse kinetic isotope effect (KIE). In contrast, STO of a 1:1 mixture of unlabeled and 5-CD3-salicylate-d8 yields a normal product isotope effect. It is proposed that aromatic and aryl-methyl hydroxylation reactions both begin with the Fe(III)-superoxo reaction with a ring carbon, yielding the inverse KIE due to sp2 → sp3 carbon hybridization. After Rieske electron transfer, the resulting Fe(III)-peroxo-salicylate intermediate can continue to aromatic hydroxylation, whereas the equivalent aryl-methyl intermediate formation must be reversible to allow the substrate exchange necessary to yield a normal product isotope effect. The resulting Fe(III)-(hydro)peroxo intermediate may be reactive or evolve through a high-valent iron intermediate to complete the aryl-methyl hydroxylation.

Nuclear Resonance Vibrational Spectroscopy Definition of Peroxy Intermediates in Catechol Dioxygenases: Factors that Determine Extra- versus Intradiol Cleavage.

The extradiol dioxygenases (EDOs) and intradiol dioxygenases (IDOs) are nonheme iron enzymes that catalyze the oxidative aromatic ring cleavage of catechol substrates, playing an essential role in the carbon cycle. The EDOs and IDOs utilize very different FeII and FeIII active sites to catalyze the regiospecificity in their catechol ring cleavage products. The factors governing this difference in cleavage have remained undefined. The EDO homoprotocatechuate 2,3-dioxygenase (HPCD) and IDO protocatechuate 3,4-dioxygenase (PCD) provide an opportunity to understand this selectivity, as key O2 intermediates have been trapped for both enzymes. Nuclear resonance vibrational spectroscopy (in conjunction with density functional theory calculations) is used to define the geometric and electronic structures of these intermediates as FeII-alkylhydroperoxo (HPCD) and FeIII-alkylperoxo (PCD) species. Critically, in both intermediates, the initial peroxo bond orientation is directed toward extradiol product formation. Reaction coordinate calculations were thus performed to evaluate both the extra- and intradiol O-O cleavage for the simple organic alkylhydroperoxo and for the FeII and FeIII metal catalyzed reactions. These results show the FeII-alkylhydroperoxo (EDO) intermediate undergoes facile extradiol O-O bond homolysis due to its extra e-, while for the FeIII-alkylperoxo (IDO) intermediate the extradiol cleavage involves a large barrier and would yield the incorrect extradiol product. This prompted our evaluation of a viable mechanism to rearrange the FeIII-alkylperoxo IDO intermediate for intradiol cleavage, revealing a key role in the rebinding of the displaced Tyr447 ligand in this rearrangement, driven by the proton delivery necessary for O-O bond cleavage.

Attitudes to reducing cervical screening frequency among UK women: A qualitative analysis.

OBJECTIVE: Cervical cancer remains a significant health threat amongst women globally despite most cervical cancers being preventable through screening and Human Papilloma Virus (HPV) vaccination. With the introduction of HPV testing and vaccination, evidence suggests that the frequency of cervical screening for women can be reduced. However, there is limited evidence on women's attitudes to possible changes to the cervical screening programme. This study explored attitudes, perceptions and beliefs of women in the United Kingdom (UK) regarding potential changes to the National Cervical Screening Programme. METHOD: Forty-four women were interviewed between November 2018 and March 2019. Reflexive thematic analysis was used to identify key themes. RESULTS: Most participants were opposed to any reduction in cervical screening frequency. Reasons included perceptions that disease would develop undetected, disempowerment, increased anxiety, reduced motivation to attend, and inefficient use of health care resources. Women perceived that they were ill-informed about the reasons for the proposed change, and that access to evidence for the basis of proposed changes may persuade them to accept reduced screening frequency. Women believed that cervical screening is a test for cancer and that HPV vaccination does not provide reliable protection against cervical cancer. These beliefs suggest that women's perception that they require more information about the basis for reduced screening frequency is correct. CONCLUSION: Any changes to cervical screening frequency need to ensure public support and trust through a robust public health initiative clearly identifying the rationale for any changes.

Advanced practice nursing initiatives in Africa, moving towards the nurse practitioner role: Experiences from the field.

AIM: This paper discusses the development and progression of the advanced practice nurse practitioner role in Africa. BACKGROUND: Providing adequate primary health care is problematic in Africa. The World Health Organization and International Council of Nurses proposed that nurses, specifically advanced practice nurse practitioners with the requisite skills in disease prevention, diagnosis and management, can be key to solving the primary care issue. SOURCES OF EVIDENCE: This paper utilized publications from PUBMED, CINAHL, policy papers, websites, workgroups, conferences, and the experiences and knowledge of authors involved in leading and moving forward key events and projects. DISCUSSION: Four African countries have formally adopted the advanced practice nurse practitioner role, with significant interest from countries throughout Africa, and ever-increasing requests for assistance regarding initiation, development and integration of advanced practice roles. Initiatives to advance the roles have been supported by the International Council of Nurses Nurse Practitioner/Advanced Practice Nurse Network and Global Academy of Research and Enterprise. Next steps and projects for future role development are discussed. CONCLUSIONS: There is a progression towards the adoption of the advanced practice nurse practitioner role in Africa, and further mechanisms are suggested to allow full uptake and utilization. IMPLICATIONS FOR NURSING PRACTICE: Prioritization and investment in initiatives implementing nurse practitioner/advanced practice nurse roles in Africa allows nurses to pursue further education, advanced role and leadership opportunities consistent with Nursing Now goals. IMPLICATIONS FOR HEALTH POLICY: Implementation of nurse practitioner/advanced practice nurse roles increases the primary care workforce, consistent with recommendations and priorities in the World Health Organization Global Strategic Directions for Nursing and Midwifery 2021-2025 helping countries ensure that nurses optimally contribute to achieving universal health coverage and other population health goals.

Dynamic Long-Range Interactions Influence Substrate Binding and Catalysis by Human Histidine Triad Nucleotide-Binding Proteins (HINTs), Key Regulators of Multiple Cellular Processes and Activators of Antiviral ProTides.

Human histidine triad nucleotide-binding (hHINT) proteins catalyze nucleotide phosphoramidase and acyl-phosphatase reactions that are essential for the activation of antiviral proTides, such as Sofosbuvir and Remdesivir. hHINT1 and hHINT2 are highly homologous but exhibit disparate roles as regulators of opioid tolerance (hHINT1) and mitochondrial activity (hHINT2). NMR studies of hHINT1 reveal a pair of dynamic surface residues (Q62, E100), which gate a conserved water channel leading to the active site 13 Å away. hHINT2 crystal structures identify analogous residues (R99, D137) and water channel. hHINT1 Q62 variants significantly alter the steady-state kcat and Km for turnover of the fluorescent substrate (TpAd), while stopped-flow kinetics indicate that KD also changes. hHINT2, like hHINT1, exhibits a burst phase of adenylation, monitored by fluorescent tryptamine release, prior to rate-limiting hydrolysis and nucleotide release. hHINT2 exhibits a much smaller burst-phase amplitude than hHINT1, which is further diminished in hHINT2 R99Q. Kinetic simulations suggest that amplitude variations can be accounted for by a variable fluorescent yield of the E·S complex from changes in the environment of bound TpAd. Isothermal titration calorimetry measurements of inhibitor binding show that these hHINT variants also alter the thermodynamic binding profile. We propose that these altered surface residues engender long-range dynamic changes that affect the orientation of bound ligands, altering the thermodynamic and kinetic characteristics of hHINT active site function. Thus, studies of the cellular roles and proTide activation potential by hHINTs should consider the importance of long-range interactions and possible protein binding surfaces far from the active site.

"A lot can happen in five years": Women's attitudes to extending cervical screening intervals.

OBJECTIVE: Evidence suggests that cervical screening intervals can be extended and lifetime cervical screening for human papillomavirus (HPV)-vaccinated women could be reduced. This study examines UK women's attitudes to extending screening intervals and assesses associations between knowledge, risk perception and HPV vaccination status, and acceptability of changes. METHODS: Using a convenience sampling strategy, an anonymous mixed-methods online survey was used and data recorded from 647 women (mean age = 28.63, SD = 8.69). RESULTS: Across the full sample, 46.1% of women indicated they would wait 5 years for their next cervical screening, while 60.2% of HPV-vaccinated women would be unwilling to have as few as three cervical screens in a lifetime. Multivariate analysis revealed those who are regular screened, those who intend to attend when invited, and those who perceive greater personal risk of cervical cancer are less likely to accept a 5-year screening interval. Qualitative findings relating to benefits of extending intervals included convenience of less tests, less physical discomfort, and psychological distress. Concerns identified included the likelihood of developing illness, increased psychological distress relating to what may be happening in the body, and worries about increased risk of cervical cancer. CONCLUSION: Women need clear and specific information about HPV timelines, their relationship with cancer risk, and the rationale for extending screening intervals.

Global emotional and spiritual well-being and resilience of Advanced Practice Nurses during the COVID-19 pandemic: A cross-sectional study.

AIM: To evaluate the impact of the COVID-19 pandemic on the emotional and spiritual well-being and resilience of a global sample of Advanced Practice Nurses. DESIGN: A web-based cross-sectional mixed methods study. Survey data were collected from Advanced Practice Nurses globally over a 2-month period ending on 31 August 2020. METHODS: The Warwick-Edinburgh Mental Well-being Scale, FACIT-12 Spiritual Well-being Scale and Connor-Davidson Resilience Scale 10 were used to quantify emotional and spiritual well-being and resilience of Advanced Practice Nurses' globally. The survey was distributed internationally using snowball sampling via a secure platform (Qualtrics). Results were analysed using various bivariate tests for associations and group differences. RESULTS: Nine hundred and twenty-eight Advanced Practice Nurses from 53 countries participated in the study. Study participants reported meaningfully lower scores in resilience and emotional well-being compared with non-pandemic scores. Participants from countries with well-developed Advanced Practice Nurses roles reported lower resilience and well-being scores compared with those from countries where Advanced Practice Nurses roles are still being developed. Each scale revealed significant positive associations with the other scales. CONCLUSIONS: Emotional and spiritual well-being and resilience of Advance Practice Nurses has been significantly impacted during the COVID-19 pandemic. Regardless of their work location, work hours, credential or demographics, the APNs in our study reported lower levels of resilience and mental well-being compared with typical scores on the instruments.

An examination of student nurse practitioners' diagnostic reasoning skills.

AIM: To examine the diagnostic reasoning skills of two nurse practitioner student cohorts. INTRODUCTION: Nurse practitioners continue to play a pivotal role in health care provision. Diagnostic reasoning is a core skill of advanced practice. A comparative diagnostic reasoning study between two student cohorts was undertaken between 2018 and 2019. METHODS: A validated diagnostic reasoning scale was completed by nurse practitioner students in the United Kingdom and the United States of America at the beginning and end of their clinical placements. The study utilized descriptive quantitative statistics from the data submitted by 22 survey respondents from an online survey. RESULTS: Analysis of the diagnostic reasoning skills suggested there was a difference in the structural memory scores when compared with the beginning of their clinical placements and the final semester of their clinical placements. CONCLUSION: Diagnostic reasoning skills are a core skill of advanced practice. As students progress through their training, these skills improve. Despite the statistical difference in diagnostic reasoning scores, caution should be made in determining any larger implications due to the small number of participants in this study. Further study is needed in this area.

Emotional well-being, spiritual well-being and resilience of advanced clinical practitioners in the United Kingdom during COVID-19: an exploratory mixed method study.

AIM: To investigate the emotional and spiritual well-being and resilience of advanced clinical practitioners during COVID. BACKGROUND: Resilience is a protective factor for emotional and spiritual well-being. The pandemic has taken a toll on health professionals due to significant physical and psychological pressures. The impact of COVID-19 on well-being and resilience of advanced clinical practitioners is not known. METHOD: Three validated scales assessed resilience, emotional and spiritual well-being. Seven hundred and thirty-four responses were analysed. RESULTS: Participants have low levels of emotional and spiritual well-being. Participants with higher levels of spirituality reported greater resilience and those with higher levels of resilience reported greater well-being. CONCLUSION: Advanced clinical practitioners' emotional and spiritual well-being and resilience has been impacted significantly during the pandemic. Interventions are needed at team, service and systems levels to enhance well-being and resilience. IMPLICATIONS FOR NURSING MANAGEMENT: Worryingly low levels of well-being and resilience in advanced clinical practitioners have been found; support to increase well-being and resilience is needed. Our findings can inform policies, resources and interventions aimed at enabling positive adaptation and enhanced resilience. Understanding and responding to the scale and impact of COVID-19 on health care workers has become a key government recommendation following the pandemic.

6-phenylpyrrolocytosine as a fluorescent probe to examine nucleotide flipping catalyzed by a DNA repair protein.

Cellular exposure to tobacco-specific nitrosamines causes formation of promutagenic O6 -[4-oxo-4-(3-pyridyl)but-1-yl]guanine (O6 -POB-G) and O6 -methylguanine (O6 -Me-G) adducts in DNA. These adducts can be directly repaired by O6 -alkylguanine-DNA alkyltransferase (AGT). Repair begins by flipping the damaged base out of the DNA helix. AGT binding and base-flipping have been previously studied using pyrrolocytosine as a fluorescent probe paired to the O6 -alkylguanine lesion, but low fluorescence yield limited the resolution of steps in the repair process. Here, we utilize the highly fluorescent 6-phenylpyrrolo-2'-deoxycytidine (6-phenylpyrrolo-C) to investigate AGT-DNA interactions. Synthetic oligodeoxynucleotide duplexes containing O6 -POB-G and O6 -Me-G adducts were placed within the CpG sites of codons 158, 245, and 248 of the p53 tumor suppressor gene and base-paired to 6-phenylpyrrolo-C in the opposite strand. Neighboring cytosine was either unmethylated or methylated. Stopped-flow fluorescence measurements were performed by mixing the DNA duplexes with C145A or R128G AGT variants. We observe a rapid, two-step, nearly irreversible binding of AGT to DNA followed by two slower steps, one of which is base-flipping. Placing 5-methylcytosine immediately 5' to the alkylated guanosine causes a reduction in rate constant of nucleotide flipping. O6 -POB-G at codon 158 decreased the base flipping rate constant by 3.5-fold compared with O6 -Me-G at the same position. A similar effect was not observed at other codons.

[The spiritual dimension in nursing]. / La dimension spirituelle dans les soins infirmiers.

Spirituality refers to that which gives us hope, meaning and purpose. It is naturally part of the holistic nature of the care provided by nurses to people going through life's challenges: health-related accident, serious and incurable illness, chronic disease, end of life, etc. A British advanced practice nurse, who has enriched her training with a doctorate in spirituality in the context of health care, enlightens this dimension, which is less formalized in France than in the United Kingdom, and suggests resources for progressing personally and professionally.

Salicylate 5-Hydroxylase: Intermediates in Aromatic Hydroxylation by a Rieske Monooxygenase.

Rieske oxygenases (ROs) catalyze a large range of oxidative chemistry. We have shown that cis-dihydrodiol-forming Rieske dioxygenases first react with their aromatic substrates via an active site nonheme Fe(III)-superoxide; electron transfer from the Rieske cluster then completes the product-forming reaction. Alternatively, two-electron-reduced Fe(III)-peroxo or hydroxo-Fe(V)-oxo activated oxygen intermediates are possible and may be utilized by other ROs to expand the catalytic range. Here, the reaction of a Rieske monooxygenase, salicylate 5-hydroxylase, that does not form a cis-dihydrodiol is examined. Single-turnover kinetic studies show fast binding of salicylate and O2. Transfer of the Rieske electron required to form the gentisate product occurs through bonds over ∼12 Å and must also be very fast. However, the observed rate constant for this reaction is much slower than expected and sensitive to substrate type. This suggests that initial reaction with salicylate occurs using the same Fe(III)-superoxo-level intermediate as Rieske dioxygenases and that this reaction limits the observed rate of electron transfer. A transient intermediate (λmax = 700 nm) with an electron paramagnetic resonance (EPR) at g = 4.3 is observed after the product is formed in the active site. The use of 17O2 (I = 5/2) results in hyperfine broadening of the g = 4.3 signal, showing that gentisate binds to the mononuclear iron via its C5-OH in the intermediate. The chromophore and EPR signal allow study of product release in the catalytic cycle. Comparison of the kinetics of single- and multiple-turnover reactions shows that re-reduction of the metal centers accelerates product release ∼300-fold, providing insight into the regulatory mechanism of ROs.

NRVS Studies of the Peroxide Shunt Intermediate in a Rieske Dioxygenase and Its Relation to the Native FeII O2 Reaction.

The Rieske dioxygenases are a major subclass of mononuclear nonheme iron enzymes that play an important role in bioremediation. Recently, a high-spin FeIII-(hydro)peroxy intermediate (BZDOp) has been trapped in the peroxide shunt reaction of benzoate 1,2-dioxygenase. Defining the structure of this intermediate is essential to understanding the reactivity of these enzymes. Nuclear resonance vibrational spectroscopy (NRVS) is a recently developed synchrotron technique that is ideal for obtaining vibrational, and thus structural, information on Fe sites, as it gives complete information on all vibrational normal modes containing Fe displacement. In this study, we present NRVS data on BZDOp and assign its structure using these data coupled to experimentally calibrated density functional theory calculations. From this NRVS structure, we define the mechanism for the peroxide shunt reaction. The relevance of the peroxide shunt to the native FeII/O2 reaction is evaluated. For the native FeII/O2 reaction, an FeIII-superoxo intermediate is found to react directly with substrate. This process, while uphill thermodynamically, is found to be driven by the highly favorable thermodynamics of proton-coupled electron transfer with an electron provided by the Rieske [2Fe-2S] center at a later step in the reaction. These results offer important insight into the relative reactivities of FeIII-superoxo and FeIII-hydroperoxo species in nonheme Fe biochemistry.

Nuclear Resonance Vibrational Spectroscopy Definition of O2 Intermediates in an Extradiol Dioxygenase: Correlation to Crystallography and Reactivity.

The extradiol dioxygenases are a large subclass of mononuclear nonheme Fe enzymes that catalyze the oxidative cleavage of catechols distal to their OH groups. These enzymes are important in bioremediation, and there has been significant interest in understanding how they activate O2. The extradiol dioxygenase homoprotocatechuate 2,3-dioxygenase (HPCD) provides an opportunity to study this process, as two O2 intermediates have been trapped and crystallographically defined using the slow substrate 4-nitrocatechol (4NC): a side-on Fe-O2-4NC species and a Fe-O2-4NC peroxy bridged species. Also with 4NC, two solution intermediates have been trapped in the H200N variant, where H200 provides a second-sphere hydrogen bond in the wild-type enzyme. While the electronic structure of these solution intermediates has been defined previously as FeIII-superoxo-catecholate and FeIII-peroxy-semiquinone, their geometric structures are unknown. Nuclear resonance vibrational spectroscopy (NRVS) is an important tool for structural definition of nonheme Fe-O2 intermediates, as all normal modes with Fe displacement have intensity in the NRVS spectrum. In this study, NRVS is used to define the geometric structure of the H200N-4NC solution intermediates in HPCD as an end-on FeIII-superoxo-catecholate and an end-on FeIII-hydroperoxo-semiquinone. Parallel calculations are performed to define the electronic structures and protonation states of the crystallographically defined wild-type HPCD-4NC intermediates, where the side-on intermediate is found to be a FeIII-hydroperoxo-semiquinone. The assignment of this crystallographic intermediate is validated by correlation to the NRVS data through computational removal of H200. While the side-on hydroperoxo semiquinone intermediate is computationally found to be nonreactive in peroxide bridge formation, it is isoenergetic with a superoxo catecholate species that is competent in performing this reaction. This study provides insight into the relative reactivities of FeIII-superoxo and FeIII-hydroperoxo intermediates in nonheme Fe enzymes and into the role H200 plays in facilitating extradiol catalysis.

Conducting research through cross national collaboration.

AIM: To explore the collaborative nature of an international research project with other advanced practice nurse researchers and critically analyse the process. BACKGROUND: Research within the nursing community is recognized internationally as important to ensure that nurses participate in cutting-edge health care and promote evidence-based practices, yet there is little detail found in literature on how a successful collaborative relationship is initiated and conducted in advanced practice research. DESIGN: Discussion paper: The purpose of this paper is to discuss the process of collaboration on a research study among advanced practice nurses from four countries who are members of an international organization. IMPLICATIONS FOR NURSING: The collaborative process in international nursing research can be challenging and rewarding. It is important to remember that there is a relationship between the complexity of the study and the time required to complete it. Keys to success include following established guidelines. CONCLUSION: This project was a valuable experience in developing collaborative relationships as well as creating partnerships for future research to build on the knowledge gained. The authors' linkages to universities facilitated their participation in the research and completion of the ethical review processes. The use of social media and university resources was indispensable.

Enzyme Substrate Complex of the H200C Variant of Homoprotocatechuate 2,3-Dioxygenase: Mössbauer and Computational Studies.

The extradiol, aromatic ring-cleaving enzyme homoprotocatechuate 2,3-dioxygenase (HPCD) catalyzes a complex chain of reactions that involve second sphere residues of the active site. The importance of the second-sphere residue His200 was demonstrated in studies of HPCD variants, such as His200Cys (H200C), which revealed significant retardations of certain steps in the catalytic process as a result of the substitution, allowing novel reaction cycle intermediates to be trapped for spectroscopic characterization. As the H200C variant largely retains the wild-type active site structure and produces the correct ring-cleaved product, this variant presents a valuable target for mechanistic HPCD studies. Here, the high-spin Fe(II) states of resting H200C and the H200C-homoprotocatechuate enzyme-substrate (ES) complex have been characterized with Mössbauer spectroscopy to assess the electronic structures of the active site in these states. The analysis reveals a high-spin Fe(II) center in a low symmetry environment that is reflected in the values of the zero-field splitting (ZFS) (D ≈ - 8 cm(-1), E/D ≈ 1/3 in ES), as well as the relative orientations of the principal axes of the (57)Fe magnetic hyperfine (A) and electric field gradient (EFG) tensors relative to the ZFS tensor axes. A spin Hamiltonian analysis of the spectra for the ES complex indicates that the magnetization axis of the integer-spin S = 2 Fe(II) system is nearly parallel to the symmetry axis, z, of the doubly occupied dxy ground orbital deduced from the EFG and A-values, an observation, which cannot be rationalized by DFT assisted crystal-field theory. In contrast, ORCA/CASSCF calculations for the ZFS tensor in combination with DFT calculations for the EFG- and A-tensors describe the experimental data remarkably well.

Structural Basis for Substrate and Oxygen Activation in Homoprotocatechuate 2,3-Dioxygenase: Roles of Conserved Active Site Histidine 200.

Kinetic and spectroscopic studies have shown that the conserved active site residue His200 of the extradiol ring-cleaving homoprotocatechuate 2,3-dioxygenase (FeHPCD) from Brevibacterium fuscum is critical for efficient catalysis. The roles played by this residue are probed here by analysis of the steady-state kinetics, pH dependence, and X-ray crystal structures of the FeHPCD position 200 variants His200Asn, His200Gln, and His200Glu alone and in complex with three catecholic substrates (homoprotocatechuate, 4-sulfonylcatechol, and 4-nitrocatechol) possessing substituents with different inductive capacity. Structures determined at 1.35-1.75 Å resolution show that there is essentially no change in overall active site architecture or substrate binding mode for these variants when compared to the structures of the wild-type enzyme and its analogous complexes. This shows that the maximal 50-fold decrease in kcat for ring cleavage, the dramatic changes in pH dependence, and the switch from ring cleavage to ring oxidation of 4-nitrocatechol by the FeHPCD variants can be attributed specifically to the properties of the altered second-sphere residue and the substrate. The results suggest that proton transfer is necessary for catalysis, and that it occurs most efficiently when the substrate provides the proton and His200 serves as a catalyst. However, in the absence of an available substrate proton, a defined proton-transfer pathway in the protein can be utilized. Changes in the steric bulk and charge of the residue at position 200 appear to be capable of altering the rate-limiting step in catalysis and, perhaps, the nature of the reactive species.

Rate-Determining Attack on Substrate Precedes Rieske Cluster Oxidation during Cis-Dihydroxylation by Benzoate Dioxygenase.

Rieske dearomatizing dioxygenases utilize a Rieske iron-sulfur cluster and a mononuclear Fe(II) located 15 Šacross a subunit boundary to catalyze O2-dependent formation of cis-dihydrodiol products from aromatic substrates. During catalysis, O2 binds to the Fe(II) while the substrate binds nearby. Single-turnover reactions have shown that one electron from each metal center is required for catalysis. This finding suggested that the reactive intermediate is Fe(III)-(H)peroxo or HO-Fe(V)═O formed by O-O bond scission. Surprisingly, several kinetic phases were observed during the single-turnover Rieske cluster oxidation. Here, the Rieske cluster oxidation and product formation steps of a single turnover of benzoate 1,2-dioxygenase are investigated using benzoate and three fluorinated analogues. It is shown that the rate constant for product formation correlates with the reciprocal relaxation time of only the fastest kinetic phase (RRT-1) for each substrate, suggesting that the slower phases are not mechanistically relevant. RRT-1 is strongly dependent on substrate type, suggesting a role for substrate in electron transfer from the Rieske cluster to the mononuclear iron site. This insight, together with the substrate and O2 concentration dependencies of RRT-1, indicates that a reactive species is formed after substrate and O2 binding but before electron transfer from the Rieske cluster. Computational studies show that RRT-1 is correlated with the electron density at the substrate carbon closest to the Fe(II), consistent with initial electrophilic attack by an Fe(III)-superoxo intermediate. The resulting Fe(III)-peroxo-aryl radical species would then readily accept an electron from the Rieske cluster to complete the cis-dihydroxylation reaction.

A Long-Lived Fe(III)-(Hydroperoxo) Intermediate in the Active H200C Variant of Homoprotocatechuate 2,3-Dioxygenase: Characterization by Mössbauer, Electron Paramagnetic Resonance, and Density Functional Theory Methods.

The extradiol-cleaving dioxygenase homoprotocatechuate 2,3-dioxygenase (HPCD) binds substrate homoprotocatechuate (HPCA) and O2 sequentially in adjacent ligand sites of the active site Fe(II). Kinetic and spectroscopic studies of HPCD have elucidated catalytic roles of several active site residues, including the crucial acid-base chemistry of His200. In the present study, reaction of the His200Cys (H200C) variant with native substrate HPCA resulted in a decrease in both kcat and the rate constants for the activation steps following O2 binding by >400 fold. The reaction proceeds to form the correct extradiol product. This slow reaction allowed a long-lived (t1/2 = 1.5 min) intermediate, H200C-HPCAInt1 (Int1), to be trapped. Mössbauer and parallel mode electron paramagnetic resonance (EPR) studies show that Int1 contains an S1 = 5/2 Fe(III) center coupled to an SR = 1/2 radical to give a ground state with total spin S = 2 (J > 40 cm(-1)) in Hexch = JS1·SR. Density functional theory (DFT) property calculations for structural models suggest that Int1 is a (HPCA semiquinone(•))Fe(III)(OOH) complex, in which OOH is protonated at the distal O and the substrate hydroxyls are deprotonated. By combining Mössbauer and EPR data of Int1 with DFT calculations, the orientations of the principal axes of the (57)Fe electric field gradient and the zero-field splitting tensors (D = 1.6 cm(-1), E/D = 0.05) were determined. This information was used to predict hyperfine splittings from bound (17)OOH. DFT reactivity analysis suggests that Int1 can evolve from a ferromagnetically coupled Fe(III)-superoxo precursor by an inner-sphere proton-coupled-electron-transfer process. Our spectroscopic and DFT results suggest that a ferric hydroperoxo species is capable of extradiol catalysis.