Obstetric violence is a misnomer.

The term "obstetric violence" has been used in the legislative language of several countries to protect mothers from abuse during pregnancy. Subsequently, it has been expanded to include a spectrum of obstetric procedures, such as induction of labor, episiotomy, and cesarean delivery, and has surfaced in the peer-reviewed literature. The term "obstetric violence" can be seen as quite strong and emotionally charged, which may lead to misunderstandings or misconceptions. It might be interpreted as implying a deliberate act of violence by healthcare providers when mistreatment can sometimes result from systemic issues, lack of training, or misunderstandings rather than intentional violence. "Obstetric mistreatment" is a more comprehensive term that can encompass a broader range of behaviors and actions. "Violence" generally refers to the intentional use of physical force to cause harm, injury, or damage to another person (eg, physical assault, domestic violence, street fights, or acts of terrorism), whereas "mistreatment" is a more general term and refers to the abuse, harm, or control exerted over another person (such as nonconsensual medical procedures, verbal abuse, disrespect, discrimination and stigmatization, or neglect, to name a few examples). There may be cases where unprofessional personnel may commit mistreatment and violence against pregnant patients, but as obstetrics is dedicated to the health and well-being of pregnant and fetal patients, mistreatment of obstetric patients should never be an intended component of professional obstetric care. It is necessary to move beyond the term "obstetric violence" in discourse and acknowledge and address the structural dimensions of abusive reproductive practices. Similarly, we do not use the term "psychiatric violence" for appropriately used professional procedures in psychiatry, such as electroshock therapy, or use the term "neurosurgical violence" when drilling a burr hole. There is an ongoing need to raise awareness about the potential mistreatment of obstetric patients within the context of abuse against women in general. Using the term "mistreatment in healthcare" instead of the more limited term "obstetric violence" is more appropriate and applies to all specialties when there is unprofessional abuse and mistreatment, such as biased care, neglect, emotional abuse (verbal), or physical abuse, including performing procedures that are unnecessary, unindicated, or without informed patient consent. Healthcare providers must promote unbiased, respectful, and patient-centered professional care; provide an ethical framework for all healthcare personnel; and work toward systemic change to prevent any mistreatment or abuse in our specialty.

Addressing challenges related to the professional practice of abortion post-Roe.

The landmark Roe vs Wade Supreme Court decision in 1973 established a constitutional right to abortion. In June 2022, the Dobbs vs Jackson Women's Health Organization Supreme Court decision brought an end to the established professional practice of abortion throughout the United States. Rights-based reductionism and zealotry threaten the professional practice of abortion. Rights-based reductionism is generally the view that moral or ethical issues can be reduced exclusively to matters of rights. In relation to abortion, there are 2 opposing forms of rights-based reductionism, namely fetal rights reductionism, which emphasizes the rights for the fetus while disregarding the rights and autonomy of the pregnant patient, and pregnant patient rights reductionism, which supports unlimited abortion without regards for the fetus. The 2 positions are irreconcilable. This article provides historical examples of the destructive nature of zealotry, which is characterized by extreme devotion to one's beliefs and an intolerant stance to opposing viewpoints, and of the importance of enlightenment to limit zealotry. This article then explores the professional responsibility model as a clinically ethically sound approach to overcome the clashing forms of rights-based reductionism and zealotry and to address the professional practice of abortion. The professional responsibility model refers to the ethical and professional obligations that obstetricians and other healthcare providers have toward pregnant patients, fetuses, and the society at large. It provides a more balanced and nuanced approach to the abortion debate, avoiding the pitfalls of reductionism and zealotry, and allows both the rights of the woman and the obligations to pregnant and fetal patients to be considered alongside broader ethical, medical, and societal implications. Constructive and respectful dialogue is crucial in addressing diverse perspectives and finding common ground. Embracing the professional responsibility model enables professionals to manage abortion responsibly, thereby prioritizing patients' interests and navigating between absolutist viewpoints to find balanced ethical solutions.

Homelessness, organ donation, transplantation, and a call for equity in the United States.

While social justice is a pillar that society seeks to uphold, in the area of organ transplantation, social justice, equity, and inclusion fail in the unbefriended and undomiciled population. Due to lack of social support of the homeless population, such status often renders these individuals ineligible to be organ recipients. Though it can be argued that organ donation by an unbefriended, undomciled patient benefits the greater good, there is clear inequity in the fact that homeless individuals are denied transplants due to inadequate social support. To illustrate such social breakdown, we describe two unbefriended, undomiciled patients brought to our hospitals by emergency services with diagnoses of intracerebral haemorrhage that progressed to brain death. This proposal represents a call to action to remediate the broken system: how the inherent inequity in organ donation by unbefriended, undomiciled patients would be ethically optimized if social support systems were implemented to allow for their candidacy for organ transplantation.

The impact of birth settings on pregnancy outcomes in the United States.

In the United States, 98.3% of patients give birth in hospitals, 1.1% give birth at home, and 0.5% give birth in freestanding birth centers. This review investigated the impact of birth settings on birth outcomes in the United States. Presently, there are insufficient data to evaluate levels of maternal mortality and severe morbidity according to place of birth. Out-of-hospital births are associated with fewer interventions such as episiotomies, epidural anesthesia, operative deliveries, and cesarean deliveries. When compared with hospital births, there are increased rates of avoidable adverse perinatal outcomes in out-of-hospital births in the United States, both for those with and without risk factors. In one recent study, the neonatal mortality rates were significantly elevated for all planned home births: 13.66 per 10,000 live births (242/177,156; odds ratio, 4.19; 95% confidence interval, 3.62-4.84; P<.0001) vs 3.27 per 10,000 live births for in-hospital Certified Nurse-Midwife-attended births (745/2,280,044; odds ratio, 1). These differences increased further when patients were stratified by recognized risk factors such as breech presentation, multiple gestations, nulliparity, advanced maternal age, and postterm pregnancy. Causes of the increased perinatal morbidity and mortality include deliveries of patients with increased risks, absence of standardized criteria to exclude high-risk deliveries, and that most midwives attending out-of-hospital births in the United States do not meet the gold standard for midwifery regulation, the International Confederation of Midwives' Global Standards for Midwifery Education. As part of the informed consent process, pregnant patients interested in out-of-hospital births should be informed of its increased perinatal risks. Hospital births should be supported for all patients, especially those with increased risks.

Anger: an underappreciated destructive force in healthcare.

Anger is an emotional state that occurs when unexpected things happen to or around oneself and is "an emotional state that varies in intensity from mild irritation to intense fury and rage." It is defined as "a strong feeling of displeasure and usually of antagonism," an emotion characterized by tension and hostility arising from frustration, real or imagined injury by another, or perceived injustice. It can manifest itself in behaviors designed to remove the object of the anger (e.g., determined action) or behaviors designed merely to express the emotion. For the Roman philosopher Seneca anger is not an uncontrollable, impulsive, or instinctive reaction. It is, rather, the cognitive assent that such initial reactions to the offending action or words are in fact unjustified. It is, rather, the cognitive assent that such initial reactions to the offending action or words are in fact unjustified. It seems that the year 2022 was a year when many Americans were plainly angry. "Why is everyone so angry?" the New York Times asked in the article "The Year We Lost It." We believe that Seneca is correct in that anger is unacceptable. Anger is a negative emotion that must be controlled, and Seneca provides us with the tools to avoid and destroy anger. Health care professionals will be more effective, content, and happier if they learn more about Seneca's writings about anger and implement his wisdom on anger from over 2000 years ago.

Identification of 2-Aryl-Quinolone Inhibitors of Cytochrome bd and Chemical Validation of Combination Strategies for Respiratory Inhibitors against Mycobacterium tuberculosis.

Mycobacterium tuberculosis cytochrome bd quinol oxidase (cyt bd), the alternative terminal oxidase of the respiratory chain, has been identified as playing a key role during chronic infection and presents a putative target for the development of novel antitubercular agents. Here, we report confirmation of successful heterologous expression of M. tuberculosis cytochrome bd. The heterologous M. tuberculosis cytochrome bd expression system was used to identify a chemical series of inhibitors based on the 2-aryl-quinolone pharmacophore. Cytochrome bd inhibitors displayed modest efficacy in M. tuberculosis growth suppression assays together with a bacteriostatic phenotype in time-kill curve assays. Significantly, however, inhibitor combinations containing our front-runner cyt bd inhibitor CK-2-63 with either cyt bcc-aa3 inhibitors (e.g., Q203) and/or adenosine triphosphate (ATP) synthase inhibitors (e.g., bedaquiline) displayed enhanced efficacy with respect to the reduction of mycobacterium oxygen consumption, growth suppression, and in vitro sterilization kinetics. In vivo combinations of Q203 and CK-2-63 resulted in a modest lowering of lung burden compared to treatment with Q203 alone. The reduced efficacy in the in vivo experiments compared to in vitro experiments was shown to be a result of high plasma protein binding and a low unbound drug exposure at the target site. While further development is required to improve the tractability of cyt bd inhibitors for clinical evaluation, these data support the approach of using small-molecule inhibitors to target multiple components of the branched respiratory chain of M. tuberculosis as a combination strategy to improve therapeutic and pharmacokinetic/pharmacodynamic (PK/PD) indices related to efficacy.

Professionally responsible coronavirus disease 2019 vaccination counseling of obstetrical and gynecologic patients.

The development of coronavirus disease 2019 vaccines in the current and planned clinical trials is essential for the success of a public health response. This paper focuses on how physicians should implement the results of these clinical trials when counseling patients who are pregnant, planning to become pregnant, breastfeeding or planning to breastfeed about vaccines with government authorization for clinical use. Determining the most effective approach to counsel patients about coronavirus disease 2019 vaccination is challenging. We address the professionally responsible counseling of 3 groups of patients-those who are pregnant, those planning to become pregnant, and those breastfeeding or planning to breastfeed. We begin with an evidence-based account of the following 5 major challenges: the limited evidence base, the documented increased risk for severe disease among pregnant coronavirus disease 2019-infected patients, conflicting guidance from government agencies and professional associations, false information about coronavirus disease 2019 vaccines, and maternal mistrust and vaccine hesitancy. We subsequently provide evidence-based, ethically justified, practical guidance for meeting these challenges in the professionally responsible counseling of patients about coronavirus disease 2019 vaccination. To guide the professionally responsible counseling of patients who are pregnant, planning to become pregnant, and breastfeeding or planning to breastfeed, we explain how obstetrician-gynecologists should evaluate the current clinical information, why a recommendation of coronavirus disease 2019 vaccination should be made, and how this assessment should be presented to patients during the informed consent process with the goal of empowering them to make informed decisions. We also present a proactive account of how to respond when patients refuse the recommended vaccination, including the elements of the legal obligation of informed refusal and the ethical obligation to ask patients to reconsider. During this process, the physician should be alert to vaccine hesitancy, ask patients to express their hesitation and reasons for it, and respectfully address them. In contrast to the conflicting guidance from government agencies and professional associations, evidence-based professional ethics in obstetrics and gynecology provides unequivocal and clear guidance: Physicians should recommend coronavirus disease 2019 vaccination to patients who are pregnant, planning to become pregnant, and breastfeeding or planning to breastfeed. To prevent widening of the health inequities, build trust in the health benefits of vaccination, and encourage coronavirus disease 2019 vaccine and treatment uptake, in addition to recommending coronavirus disease 2019 vaccinations, physicians should engage with communities to tailor strategies to overcome mistrust and deliver evidence-based information, robust educational campaigns, and novel approaches to immunization.

Rational Design, Synthesis, and Biological Evaluation of Heterocyclic Quinolones Targeting the Respiratory Chain of Mycobacterium tuberculosis.

A high-throughput screen (HTS) was undertaken against the respiratory chain dehydrogenase component, NADH:menaquinone oxidoreductase (Ndh) of Mycobacterium tuberculosis (Mtb). The 11000 compounds were selected for the HTS based on the known phenothiazine Ndh inhibitors, trifluoperazine and thioridazine. Combined HTS (11000 compounds) and in-house screening of a limited number of quinolones (50 compounds) identified ∼100 hits and four distinct chemotypes, the most promising of which contained the quinolone core. Subsequent Mtb screening of the complete in-house quinolone library (350 compounds) identified a further ∼90 hits across three quinolone subtemplates. Quinolones containing the amine-based side chain were selected as the pharmacophore for further modification, resulting in metabolically stable quinolones effective against multi drug resistant (MDR) Mtb. The lead compound, 42a (MTC420), displays acceptable antituberculosis activity (Mtb IC50 = 525 nM, Mtb Wayne IC50 = 76 nM, and MDR Mtb patient isolates IC50 = 140 nM) and favorable pharmacokinetic and toxicological profiles.

Antitubercular pharmacodynamics of phenothiazines.

OBJECTIVES: Phenothiazines have been shown to exhibit in vitro and in vivo activity against Mycobacterium tuberculosis (Mtb) and multidrug-resistant Mtb. They are predicted to target the genetically validated respiratory chain component type II NADH:quinone oxidoreductase (Ndh). Using a set of compounds containing the phenothiazine pharmacophore, we have (i) investigated whether chemical validation data support the molecular target and (ii) evaluated pharmacophore tractability for further drug development. METHODS: Recombinant Mtb Ndh was generated and its functionality confirmed by steady-state kinetics. Pharmacodynamic profiling of the phenothiazines, including antitubercular efficacy in aerobic and O2-limited conditions, time-kill assays and isobole analyses against first-line antituberculars, was performed. Potential mitochondrial toxicity was assessed in a modified HepG2 cell-line assay and against bovine cytochrome bc1. RESULTS: Steady-state kinetic analyses revealed a substrate preference for coenzyme Q2 and an inability to utilize NADPH. A positive correlation between recombinant Ndh inhibition and kill of aerobically cultured Mtb was observed, whilst enhanced potency was demonstrated in a hypoxic model. Time-kill studies revealed the phenothiazines to be bactericidal whilst isobolograms exposed antagonism with isoniazid, indicative of intracellular NADH/NAD(+) couple perturbation. At therapeutic levels, phenothiazine-mediated toxicity was appreciable; however, specific mitochondrial targeting was excluded. CONCLUSIONS: Data generated support the hypothesis that Ndh is the molecular target of phenothiazines. The favourable pharmacodynamic properties of the phenothiazines are consistent with a target product profile that includes activity against dormant/persistent bacilli, rapid bactericidal activity and activity against drug-resistant Mtb by a previously unexploited mode of action. These properties warrant further medicinal chemistry to improve potency and safety.

Generation of quinolone antimalarials targeting the Plasmodium falciparum mitochondrial respiratory chain for the treatment and prophylaxis of malaria.

There is an urgent need for new antimalarial drugs with novel mechanisms of action to deliver effective control and eradication programs. Parasite resistance to all existing antimalarial classes, including the artemisinins, has been reported during their clinical use. A failure to generate new antimalarials with novel mechanisms of action that circumvent the current resistance challenges will contribute to a resurgence in the disease which would represent a global health emergency. Here we present a unique generation of quinolone lead antimalarials with a dual mechanism of action against two respiratory enzymes, NADH:ubiquinone oxidoreductase (Plasmodium falciparum NDH2) and cytochrome bc(1). Inhibitor specificity for the two enzymes can be controlled subtly by manipulation of the privileged quinolone core at the 2 or 3 position. Inhibitors display potent (nanomolar) activity against both parasite enzymes and against multidrug-resistant P. falciparum parasites as evidenced by rapid and selective depolarization of the parasite mitochondrial membrane potential, leading to a disruption of pyrimidine metabolism and parasite death. Several analogs also display activity against liver-stage parasites (Plasmodium cynomolgi) as well as transmission-blocking properties. Lead optimized molecules also display potent oral antimalarial activity in the Plasmodium berghei mouse malaria model associated with favorable pharmacokinetic features that are aligned with a single-dose treatment. The ease and low cost of synthesis of these inhibitors fulfill the target product profile for the generation of a potent, safe, and inexpensive drug with the potential for eventual clinical deployment in the control and eradication of falciparum malaria.

Identification of novel antimalarial chemotypes via chemoinformatic compound selection methods for a high-throughput screening program against the novel malarial target, PfNDH2: increasing hit rate via virtual screening methods.

Malaria is responsible for approximately 1 million deaths annually; thus, continued efforts to discover new antimalarials are required. A HTS screen was established to identify novel inhibitors of the parasite's mitochondrial enzyme NADH:quinone oxidoreductase (PfNDH2). On the basis of only one known inhibitor of this enzyme, the challenge was to discover novel inhibitors of PfNDH2 with diverse chemical scaffolds. To this end, using a range of ligand-based chemoinformatics methods, ~17000 compounds were selected from a commercial library of ~750000 compounds. Forty-eight compounds were identified with PfNDH2 enzyme inhibition IC(50) values ranging from 100 nM to 40 µM and also displayed exciting whole cell antimalarial activity. These novel inhibitors were identified through sampling 16% of the available chemical space, while only screening 2% of the library. This study confirms the added value of using multiple ligand-based chemoinformatic approaches and has successfully identified novel distinct chemotypes primed for development as new agents against malaria.

Identification, design and biological evaluation of bisaryl quinolones targeting Plasmodium falciparum type II NADH:quinone oxidoreductase (PfNDH2).

A program was undertaken to identify hit compounds against NADH:ubiquinone oxidoreductase (PfNDH2), a dehydrogenase of the mitochondrial electron transport chain of the malaria parasite Plasmodium falciparum. PfNDH2 has only one known inhibitor, hydroxy-2-dodecyl-4-(1H)-quinolone (HDQ), and this was used along with a range of chemoinformatics methods in the rational selection of 17 000 compounds for high-throughput screening. Twelve distinct chemotypes were identified and briefly examined leading to the selection of the quinolone core as the key target for structure-activity relationship (SAR) development. Extensive structural exploration led to the selection of 2-bisaryl 3-methyl quinolones as a series for further biological evaluation. The lead compound within this series 7-chloro-3-methyl-2-(4-(4-(trifluoromethoxy)benzyl)phenyl)quinolin-4(1H)-one (CK-2-68) has antimalarial activity against the 3D7 strain of P. falciparum of 36 nM, is selective for PfNDH2 over other respiratory enzymes (inhibitory IC(50) against PfNDH2 of 16 nM), and demonstrates low cytotoxicity and high metabolic stability in the presence of human liver microsomes. This lead compound and its phosphate pro-drug have potent in vivo antimalarial activity after oral administration, consistent with the target product profile of a drug for the treatment of uncomplicated malaria. Other quinolones presented (e.g., 6d, 6f, 14e) have the capacity to inhibit both PfNDH2 and P. falciparum cytochrome bc(1), and studies to determine the potential advantage of this dual-targeting effect are in progress.

Identification, design and biological evaluation of heterocyclic quinolones targeting Plasmodium falciparum type II NADH:quinone oxidoreductase (PfNDH2).

Following a program undertaken to identify hit compounds against NADH:ubiquinone oxidoreductase (PfNDH2), a novel enzyme target within the malaria parasite Plasmodium falciparum, hit to lead optimization led to identification of CK-2-68, a molecule suitable for further development. In order to reduce ClogP and improve solubility of CK-2-68 incorporation of a variety of heterocycles, within the side chain of the quinolone core, was carried out, and this approach led to a lead compound SL-2-25 (8b). 8b has IC(50)s in the nanomolar range versus both the enzyme and whole cell P. falciparum (IC(50) = 15 nM PfNDH2; IC(50) = 54 nM (3D7 strain of P. falciparum) with notable oral activity of ED(50)/ED(90) of 1.87/4.72 mg/kg versus Plasmodium berghei (NS Strain) in a murine model of malaria when formulated as a phosphate salt. Analogues in this series also demonstrate nanomolar activity against the bc(1) complex of P. falciparum providing the potential added benefit of a dual mechanism of action. The potent oral activity of 2-pyridyl quinolones underlines the potential of this template for further lead optimization studies.

Characterization of Cupriavidus metallidurans CYP116B1--a thiocarbamate herbicide oxygenating P450-phthalate dioxygenase reductase fusion protein.

The novel cytochrome P450/redox partner fusion enzyme CYP116B1 from Cupriavidus metallidurans was expressed in and purified from Escherichia coli. Isolated CYP116B1 exhibited a characteristic Fe(II)CO complex with Soret maximum at 449 nm. EPR and resonance Raman analyses indicated low-spin, cysteinate-coordinated ferric haem iron at both 10 K and ambient temperature, respectively, for oxidized CYP116B1. The EPR of reduced CYP116B1 demonstrated stoichiometric binding of a 2Fe-2S cluster in the reductase domain. FMN binding in the reductase domain was confirmed by flavin fluorescence studies. Steady-state reduction of cytochrome c and ferricyanide were supported by both NADPH/NADH, with NADPH used more efficiently (K(m[NADPH]) = 0.9 ± 0.5 µM and K(m[NADH]) = 399.1 ± 52.1 µM). Stopped-flow studies of NAD(P)H-dependent electron transfer to the reductase confirmed the preference for NADPH. The reduction potential of the P450 haem iron was -301 ± 7 mV, with retention of haem thiolate ligation in the ferrous enzyme. Redox potentials for the 2Fe-2S and FMN cofactors were more positive than that of the haem iron. Multi-angle laser light scattering demonstrated CYP116B1 to be monomeric. Type I (substrate-like) binding of selected unsaturated fatty acids (myristoleic, palmitoleic and arachidonic acids) was shown, but these substrates were not oxidized by CYP116B1. However, CYP116B1 catalysed hydroxylation (on propyl chains) of the herbicides S-ethyl dipropylthiocarbamate (EPTC) and S-propyl dipropylthiocarbamate (vernolate), and the subsequent N-dealkylation of vernolate. CYP116B1 thus has similar thiocarbamate-oxidizing catalytic properties to Rhodoccocus erythropolis CYP116A1, a P450 involved in the oxidative degradation of EPTC.

Cytochrome b mutation Y268S conferring atovaquone resistance phenotype in malaria parasite results in reduced parasite bc1 catalytic turnover and protein expression.

Atovaquone is an anti-malarial drug used in combination with proguanil (e.g. Malarone(TM)) for the curative and prophylactic treatment of malaria. Atovaquone, a 2-hydroxynaphthoquinone, is a competitive inhibitor of the quinol oxidation (Q(o)) site of the mitochondrial cytochrome bc(1) complex. Inhibition of this enzyme results in the collapse of the mitochondrial membrane potential, disruption of pyrimidine biosynthesis, and subsequent parasite death. Resistance to atovaquone in the field is associated with point mutations in the Q(o) pocket of cytochrome b, most notably near the conserved Pro(260)-Glu(261)-Trp(262)-Tyr(263) (PEWY) region in the ef loop). The effect of this mutation has been extensively studied in model organisms but hitherto not in the parasite itself. Here, we have performed a molecular and biochemical characterization of an atovaquone-resistant field isolate, TM902CB. Molecular analysis of this strain reveals the presence of the Y268S mutation in cytochrome b. The Y268S mutation is shown to confer a 270-fold shift of the inhibitory constant (K(i)) for atovaquone with a concomitant reduction in the V(max) of the bc(1) complex of ∼40% and a 3-fold increase in the observed K(m) for decylubiquinol. Western blotting analyses reveal a reduced iron-sulfur protein content in Y268S bc(1) suggestive of a weakened interaction between this subunit and cytochrome b. Gene expression analysis of the TM902CB strain reveals higher levels of expression, compared with the 3D7 (atovaquone-sensitive) control strain in bc(1) and cytochrome c oxidase genes. It is hypothesized that the observed differential expression of these and other key genes offsets the fitness cost resulting from reduced bc(1) activity.

Transforming rhinacanthin analogues from potent anticancer agents into potent antimalarial agents.

Twenty-six novel naphthoquinone aliphatic esters were synthesized by esterification of 1,4-naphthoquinone alcohols with various aliphatic acids. The 1,4-naphthoquinone alcohols were prepared from 1-hydroxy-2-naphthoic acid in nine steps with excellent yields. Twenty-four of the novel synthetic naphthoquinone esters showed significant antimalarial activity with IC(50) values in the range of 0.03-16.63 microM. The length of the aliphatic chain and the presence of C-2' substituents on the propyl chain affected the activity. Interestingly, compounds 31 and 37 showed very good antimalarial activity and were not toxic to normal Vero cells, and the PTI values of 31 (>1990.38) and 37 (1825.94) are excellent. Both 31 and 37 showed potent inhibition against P. falciparum 3D7 cyt bc(1) and no inhibition on rat cyt bc(1). They showed IC(50) values in the nanomolar range, providing full inhibition of cyt bc(1) with one molecule inhibitor bound per cyt bc(1) monomer at the Q(o) site.

Chapter 17 Type II NADH: quinone oxidoreductases of Plasmodium falciparum and Mycobacterium tuberculosis kinetic and high-throughput assays.

Type II NADH: quinone oxidoreductases (ndh) are flavoenzymes found in a broad range of organisms including plants, fungi, protozoa, and bacteria. The ndh enzymes catalyze the oxidation of NADH with concomitant reduction of quinone (Q). These membrane-bound respiratory enzymes differ from the canonical NADH: dehydrogenase (complex I), because they are not involved in the vectorial transfer of protons across membranes. In Plasmodium falciparum and Mycobacterium tuberculosis, causative agents of malaria and tuberculosis, respectively, ndhs have aroused interest because of the essential role played in maintaining a reduced Q-pool during infection. In this chapter, we present methods for the measurement of steady-state parameters for ndhs from both pathogens, highlighting best practices and caveats. In addition, owing to the interest in ndhs as potential chemotherapeutic targets, we describe a miniaturized endpoint assay that is validated for high-throughput screening (HTS) of chemical libraries.

Biophysical characterization of the sterol demethylase P450 from Mycobacterium tuberculosis, its cognate ferredoxin, and their interactions.

Mycobacterium tuberculosis encodes a P450 of the sterol demethylase family (CYP51) chromosomally located adjacent to a ferredoxin (Fdx). CYP51 and Fdx were purified to homogeneity and characterized. Spectroscopic analyses were consistent with cysteinate- and aqua-ligated heme iron in CYP51. An epsilon419 of 134 mM(-1) cm(-1) was determined for oxidized CYP51. Analysis of interactions of 1-, 2-, and 4-phenylimidazoles with CYP51 showed that the 1- and 4-forms were heme iron-coordinating inhibitors, while 2-phenylimidazole induced a substrate-like optical shift. The 2-phenyimidazole-bound CYP51 demonstrated unusual decreases in high-spin heme iron content at elevated temperatures and an almost complete absence of high-spin heme iron by low-temperature EPR. These data suggest thermally induced alterations in CYP51 active site structure and/or binding modes for the small ligand. Reduction of CYP51 in the presence of carbon monoxide leads to formation of an Fe(II)-CO complex with a Soret absorption maximum at 448.5 nm, which collapses (at 0.246 min(-1) at pH 7.0) forming a species with a Soret maximum at 421.5 nm (the inactive P420 form). The rate of P420 formation is accelerated at lower pH, consistent with protonation of the cysteinate (Cys 394) to a thiol underlying the P450-P420 transition. The P450 form is stabilized by estriol, which induces a type I spectral shift on binding CYP51 (Kd = 21.7 microM). Nonstandard spectral changes occur on CYP51 reduction (using either dithionite or natural redox partners), including a blue-shifted Soret band and development of a strong feature at approximately 558.5 nm, suggestive of cysteine thiol ligation. Thus, ligand-free ferrous CYP51 is prone to thiolate ligand protonation even in the absence of carbon monoxide. Analysis of reoxidized CYP51 demonstrates that the enzyme re-forms P450, indicating that Cys 394 thiol is readily deprotonated to thiolate in the ferric form. Spectroscopic analysis of Fdx by EPR (resonance at g = 2.03) and magnetic CD (intensity for oxidized and reduced forms and signal intensity dependence on field strength and temperature) demonstrated that Fdx binds a [3Fe-4S] iron-sulfur cluster. Potentiometric studies show that the midpoint potential for ligand-free CYP51 is -375 mV, increasing to -225 mV in the estriol-bound form. The Fdx potential is -31 mV. Fdx forms a productive electron transfer complex with CYP51 and reduces it at a rate of 3.0 min(-1) in the ligand-free form and 4.3 min(-1) in the estriol-bound form, despite a thermodynamic barrier. Steady-state analysis of a M. tuberculosis class I redox system comprising flavoprotein reductase A (FprA), Fdx, and estriol-bound CYP51 indicates heme iron reduction as a rate-limiting step.