Cryo-EM structures of Escherichia coli cytochrome bo3 reveal bound phospholipids and ubiquinone-8 in a dynamic substrate binding site.

Two independent structures of the proton-pumping, respiratory cytochrome bo3 ubiquinol oxidase (cyt bo3 ) have been determined by cryogenic electron microscopy (cryo-EM) in styrene-maleic acid (SMA) copolymer nanodiscs and in membrane scaffold protein (MSP) nanodiscs to 2.55- and 2.19-Å resolution, respectively. The structures include the metal redox centers (heme b, heme o3 , and CuB), the redox-active cross-linked histidine-tyrosine cofactor, and the internal water molecules in the proton-conducting D channel. Each structure also contains one equivalent of ubiquinone-8 (UQ8) in the substrate binding site as well as several phospholipid molecules. The isoprene side chain of UQ8 is clamped within a hydrophobic groove in subunit I by transmembrane helix TM0, which is only present in quinol oxidases and not in the closely related cytochrome c oxidases. Both structures show carbonyl O1 of the UQ8 headgroup hydrogen bonded to D75I and R71I In both structures, residue H98I occupies two conformations. In conformation 1, H98I forms a hydrogen bond with carbonyl O4 of the UQ8 headgroup, but in conformation 2, the imidazole side chain of H98I has flipped to form a hydrogen bond with E14I at the N-terminal end of TM0. We propose that H98I dynamics facilitate proton transfer from ubiquinol to the periplasmic aqueous phase during oxidation of the substrate. Computational studies show that TM0 creates a channel, allowing access of water to the ubiquinol headgroup and to H98I.

Activation of NF-κB and p300/CBP potentiates cancer chemoimmunotherapy through induction of MHC-I antigen presentation.

Many cancers evade immune rejection by suppressing major histocompatibility class I (MHC-I) antigen processing and presentation (AgPP). Such cancers do not respond to immune checkpoint inhibitor therapies (ICIT) such as PD-1/PD-L1 [PD-(L)1] blockade. Certain chemotherapeutic drugs augment tumor control by PD-(L)1 inhibitors through potentiation of T-cell priming but whether and how chemotherapy enhances MHC-I-dependent cancer cell recognition by cytotoxic T cells (CTLs) is not entirely clear. We now show that the lysine acetyl transferases p300/CREB binding protein (CBP) control MHC-I AgPPM expression and neoantigen amounts in human cancers. Moreover, we found that two distinct DNA damaging drugs, the platinoid oxaliplatin and the topoisomerase inhibitor mitoxantrone, strongly up-regulate MHC-I AgPP in a manner dependent on activation of nuclear factor kappa B (NF-κB), p300/CBP, and other transcription factors, but independently of autocrine IFNγ signaling. Accordingly, NF-κB and p300 ablations prevent chemotherapy-induced MHC-I AgPP and abrogate rejection of low MHC-I-expressing tumors by reinvigorated CD8+ CTLs. Drugs like oxaliplatin and mitoxantrone may be used to overcome resistance to PD-(L)1 inhibitors in tumors that had "epigenetically down-regulated," but had not permanently lost MHC-I AgPP activity.

Disruption of zinc (II) binding and dimeric protein structure of the XIAP-RING domain by copper (I) ions.

Modulation of metalloprotein structure and function via metal ion substitution may constitute a molecular basis for metal ion toxicity and/or metal-mediated functional control. The X-linked Inhibitor of Apoptosis Protein (XIAP) is a metalloprotein that requires zinc for proper structure and function. In addition to its role as a modulator of apoptosis, XIAP has been implicated in copper homeostasis. Given the similar coordination preferences of copper and zinc, investigation of XIAP structure and function upon interaction with copper is relevant. The Really Interesting New Gene (RING) domain of XIAP is representative of a class of zinc finger proteins that utilize a bi-nuclear zinc-binding motif to maintain proper structure and ubiquitin ligase function. Herein, we report the characterization of copper (I) binding to the Zn2-RING domain of XIAP. Electronic absorption studies that monitor copper-thiolate interactions demonstrate that the RING domain of XIAP binds 5-6 Cu(I) ions and that copper is thermodynamically preferred relative to zinc. Repetition of the experiments in the presence of the Zn(II)-specific dye Mag-Fura2 shows that Cu(I) addition results in Zn(II) ejection from the protein, even in the presence of glutathione. Loss of dimeric structure of the RING domain, which is a requirement for its ubiquitin ligase activity, upon copper substitution at the zinc-binding sites, was readily observed via size exclusion chromatography. These results provide a molecular basis for the modulation of RING function by copper and add to the growing body of literature that describe the impact of Cu(I) on zinc metalloprotein structure and function.

USPSTF found insufficient evidence on the benefits and harms of screening adults ≥50 y for AF.

SOURCE CITATION: Davidson KW, Barry MJ, Mangione CM, et al. Screening for atrial fibrillation: US Preventive Services Task Force recommendation statement. JAMA. 2022;327:360-7. 35076659.

Searching for the low affinity ubiquinone binding site in cytochrome bo3 from Escherichia coli.

The cytochrome bo3 ubiquinol oxidase is one of three respiratory oxygen reductases in the aerobic respiratory chain of Escherichia coli. The generally accepted model of catalysis assumes that cyt bo3 contains two distinct ubiquinol binding sites: (i) a low affinity (QL) site which is the traditional substrate binding site; and (ii) a high affinity (QH) site where a "permanently" bound quinone acts as a cofactor, taking two electrons from the substrate quinol and passing them one-by-one to the heme b component of the enzyme which, in turn, transfers them to the heme o3/CuB active site. Whereas the residues at the QH site are well defined, the location of the QL site remains unknown. The published X-ray structure does not contain quinone, and substantial amounts of the protein are missing as well. A recent bioinformatics study by Bossis et al. [Biochem J. (2014) 461, 305-314] identified a sequence motif G163EFX3GWX2Y173 as the likely QL site in the family of related quinol oxidases. In the current work, this was tested by site-directed mutagenesis. The results show that these residues are not important for catalytic function and do not define the QL substrate binding site.

Location of the Substrate Binding Site of the Cytochrome bo3 Ubiquinol Oxidase from Escherichia coli.

Cytochrome bo3 is a respiratory proton-pumping oxygen reductase that is a member of the heme-copper superfamily that utilizes ubiquinol-8 (Q8H2) as a substrate. The current consensus model has Q8H2 oxidized at a low affinity site (QL), passing electrons to a tightly bound quinone cofactor at a high affinity site (QH site) that stabilizes the one-electron reduced ubisemiquinone, facilitating the transfer of electrons to the redox active metal centers where O2 is reduced to water. The current work shows that the Q8 bound to the QH site is more dynamic than previously thought. In addition, mutations of residues at the QH site that do not abolish activity have been re-examined and shown to have properties expected of mutations at the substrate binding site (QL): an increase in the KM of the substrate ubiquinol-1 (up to 4-fold) and an increase in the apparent Ki of the inhibitor HQNO (up to 8-fold). The data suggest that there is only one binding site for ubiquinol in cyt bo3 and that site corresponds to the QH site.

Exploring the proton pump and exit pathway for pumped protons in cytochrome ba3 from Thermus thermophilus.

The heme-copper oxygen reductases are redox-driven proton pumps. In the current work, the effects of mutations in a proposed exit pathway for pumped protons are examined in the ba(3)-type oxygen reductase from Thermus thermophilus, leading from the propionates of heme a(3) to the interface between subunits I and II. Recent studies have proposed important roles for His376 and Asp372, both of which are hydrogen-bonded to propionate-A of heme a(3), and for Glu126(II) (subunit II), which is hydrogen-bonded to His376. Based on the current results, His376, Glu126(II), and Asp372 are not essential for either oxidase activity or proton pumping. In addition, Tyr133, which is hydrogen-bonded to propionate-D of heme a(3), was also shown not to be essential for function. However, two mutations of the residues hydrogen-bonded to propionate-A, Asp372Ile and His376Asn, retain high electron transfer activity and normal spectral features but, in different preparations, either do not pump protons or exhibit substantially diminished proton pumping. It is concluded that either propionate-A of heme a(3) or possibly the cluster of groups centered about the conserved water molecule that hydrogen-bonds to both propionates-A and -D of heme a(3) is a good candidate to be the proton loading site.

Kinetics and intermediates of the reaction of fully reduced Escherichia coli bo3 ubiquinol oxidase with O2.

Cytochrome bo3 ubiquinol oxidase from Escherichia coli catalyzes the reduction of O2 to water by ubiquinol. The reaction mechanism and the role of ubiquinol continue to be a subject of discussion. In this study, we report a detailed kinetic scheme of the reaction of cytochrome bo3 with O2 with steps specific to ubiquinol. The reaction was investigated using the CO flow-flash method, and time-resolved optical absorption difference spectra were collected from 1 µs to 20 ms after photolysis. Singular value decomposition-based global exponential fitting resolved five apparent lifetimes, 22 µs, 30 µs, 42 µs, 470 µs, and 2.0 ms. The reaction mechanism was derived by an algebraic kinetic analysis method using frequency-shifted spectra of known bovine states to identify the bo3 intermediates. It shows 42 µs O2 binding (3.8 × 10(7) M(-1) s(-1)), producing compound A, followed by faster (22 µs) heme b oxidation, yielding a mixture of PR and F, and rapid heme b rereduction by ubiquinol (30 µs), producing the F intermediate and semiquinone. In the 470 µs step, the o3 F state is converted into the o3(3+) oxidized state, presumably by semiquinone/ubiquinol, without the concomitant oxidation of heme b. The final 2 ms step shows heme b reoxidation and the partial rereduction of the binuclear center and, following O2 binding, the formation of a mixture of P and F during a second turnover cycle. The results show that ubiquinol/semiquinone plays a complex role in the mechanism of O2 reduction by bo3, displaying kinetic steps that have no analogy in the CuA-containing heme-copper oxidases.

Evidence for distinct electron transfer processes in terminal oxidases from different origin by means of protein film voltammetry.

Cytochrome aa3 from Paracoccus denitrificans and cytochrome ba3 from Thermus thermophilus, two distinct members of the heme-copper oxidase superfamily, were immobilized on electrodes modified with gold nanoparticles. This procedure allowed us to achieve direct electron transfer between the enzyme and the gold nanoparticles and to obtain evidence for different electrocatalytic properties of the two enzymes. The pH dependence and thermostability reveal that the enzymes are highly adapted to their native environments. These results suggest that evolution resulted in different solutions to the common problem of electron transfer to oxygen.

Direct Electrochemistry of Cytochrome bo Oxidase at a series of Gold Nanoparticles-Modified Electrodes.

New membrane-protein based electrodes were prepared incorporating cytochrome bo(3) from E. coli and gold nanoparticles. Direct electron transfer between the electrode and the immobilized enzymes was achieved, resulting in an electrocatalytic activity in presence of O(2). The size of the gold nanoparticles was shown to be important and smaller particles were shown to reduce the overpotential of the process.

Atrial fibrillation and stroke.

INTRODUCTION: Stroke is one of the leading causes of mortality and morbidity globally. Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia. It is set to reach epidemic proportions. AF is associated with a five-fold increase in risk of stroke. Strokes caused by AF more often are fatal or result in severe disability. Even though the incidence of stroke has been significantly reduced by oral anticoagulation, AF is thought to account for a significant proportion of cryptogenic strokes where no etiology is identified. AREAS COVERED: This article reviews the literature related to AF and stroke, pathophysiological insights, diagnosis of AF in stroke patients, and its management (Graphical Abstract). EXPERT OPINION: The pathophysiology of thrombogenesis that links AF and stroke is not well understood and is an area of active research to identify new therapeutic targets to prevent AF and stroke. As the nature of AF and stroke is multifaceted, an integrated care approach to managing AF and stroke is increasingly essential.

Interactions of intermediate semiquinone with surrounding protein residues at the Q(H) site of wild-type and D75H mutant cytochrome bo3 from Escherichia coli.

Selective (15)N isotope labeling of the cytochrome bo(3) ubiquinol oxidase from Escherichia coli with auxotrophs was used to characterize the hyperfine couplings with the side-chain nitrogens from residues R71, H98, and Q101 and peptide nitrogens from residues R71 and H98 around the semiquinone (SQ) at the high-affinity Q(H) site. The two-dimensional ESEEM (HYSCORE) data have directly identified N(ε) of R71 as an H-bond donor carrying the largest amount of unpaired spin density. In addition, weaker hyperfine couplings with the side-chain nitrogens from all residues around the SQ were determined. These hyperfine couplings reflect a distribution of the unpaired spin density over the protein in the SQ state of the Q(H) site and the strength of interaction with different residues. The approach was extended to the virtually inactive D75H mutant, where the intermediate SQ is also stabilized. We found that N(ε) of a histidine residue, presumably H75, carries most of the unpaired spin density instead of N(ε) of R71, as in wild-type bo(3). However, the detailed characterization of the weakly coupled (15)N atoms from selective labeling of R71 and Q101 in D75H was precluded by overlap of the (15)N lines with the much stronger ~1.6 MHz line from the quadrupole triplet of the strongly coupled (14)N(ε) atom of H75. Therefore, a reverse labeling approach, in which the enzyme was uniformly labeled except for selected amino acid types, was applied to probe the contribution of R71 and Q101 to the (15)N signals. Such labeling has shown only weak coupling with all nitrogens of R71 and Q101. We utilize density functional theory-based calculations to model the available information about (1)H, (15)N, and (13)C hyperfine couplings for the Q(H) site and to describe the protein-substrate interactions in both enzymes. In particular, we identify the factors responsible for the asymmetric distribution of the unpaired spin density and ponder the significance of this asymmetry to the quinone's electron transfer function.

The three-spin intermediate at the O-O cleavage and proton-pumping junction in heme-Cu oxidases.

Understanding the mechanistic coupling of molecular oxygen reduction and proton pumping for adenosine triphosphate synthesis during cellular respiration is the primary goal of research on heme-copper oxidases­the terminal complex in the membrane-bound electron transport chain. Cleavage of the oxygen-oxygen bond by the heme-copper oxidases forms the key intermediate PM, which initiates proton pumping. This intermediate is now experimentally defined by variable-temperature, variable-field magnetic circular dichroism spectroscopy on a previously unobserved excited state feature associated with its heme iron(IV)-oxo center. These data provide evidence that the iron(IV)-oxo in PM is magnetically coupled to both a copper(II) and a cross-linked tyrosyl radical in the active site. These results provide new insight into the oxygen-oxygen bond cleavage and proton-pumping mechanisms of heme-copper oxidases.

Better Together: A Mixed-Methods Study of Palliative Care Co-Management for Patients with Interstitial Lung Disease.

Background: The morbidity and mortality of interstitial lung disease (ILD) is high, despite novel therapeutics. Recognizing unmet needs for symptom management, advance care planning (ACP), and support for people with ILD and their families, we developed a palliative care-ILD collaborative care pilot program to improve access to palliative care. Methods: In the quantitative arm of this mixed-methods study, we evaluated which patients were cared for through the palliative care co-management program and the impact of the program on rates of ACP and opioid prescribing. In the qualitative arm, we interviewed patients and family caregivers, as well as pulmonary and palliative care clinicians, to understand perceptions about palliative care. Results: Thirty-one patients were co-managed by the palliative care and ILD teams during the study period. Half (48.4%) had idiopathic pulmonary fibrosis. Mean forced vital capacity (FVC) was 61.7%. Nearly half (48.4%) received all of their palliative care via telehealth. With palliative care, the rate of ACP notes increased from 3.2% to 100% (p < 0.001), rate of advance directive completion increased from 22.6% to 35.5% (p = 0.046), and rate of physician orders for life-sustaining treatments (POLST) form completion increased from 0% to 35.5% (p = 0.001). Half (51.6%) were prescribed opiates, overwhelmingly short-acting opiates to use as needed for severe episodic dyspnea. Themes from the qualitative analyses included that the palliative care team was supportive and patient-centered, improved symptoms and medication side effects, and enhanced illness understanding. Clinicians reported how palliative care co-management improved patient care and clinician experience, but barriers to referral remain including misperceptions about palliative care on the part of providers and patients. Conclusions: Palliative care co-management for patients with moderately severe ILD holds promise, and our experience can inform groups at other centers who are interested in developing such care models. Ongoing challenges include systematically reaching all patients who are likely to benefit.

Experiential Communications Curriculum to Improve Resident Preparedness When Responding to Discriminatory Comments in the Workplace.

BACKGROUND: Patients and families can make discriminatory comments leading to physician distress. Residents receive little training in appropriate responses to such comments and may be ill equipped to respond to intolerance without alienating the individual(s) making the comments. OBJECTIVE: We assessed whether a simulated curriculum would enhance pediatrics residents' ability to effectively respond to discriminatory comments. METHODS: In the 2016-2017 academic year, we modified an existing communication skills curriculum for senior pediatrics residents. Residents engaged a simulated parent who used discriminatory speech in 4 scenarios, followed by a group debriefing. We conducted anonymous surveys to assess residents' preparedness to respond to these comments before and immediately following participation and examined their experience with discriminatory comments in the workplace. RESULTS: The majority of residents reported prior experience with discriminatory comments (32 of 45 [71%] witnessed such comments, and 27 of 48 [56%] were targeted by such comments), most often regarding age, race, and ethnicity. Mean precourse scores ranged from 2.1 to 3.1 (on a 5-point scale) regarding ability to engage in a firm yet respectful dialogue, to reference the hospital code of conduct, to coach a learner to respond, and to facilitate a team debrief. Mean postcourse scores improved significantly for these questions (range 3.8-4.1). The greatest improvement was in referencing the code of conduct (2.1 versus 4.0, P < .001). CONCLUSIONS: Immediately after participating in simulation, pediatrics residents reported a significant improvement in self-reported readiness to respond to discriminatory comments made by a parent and reported the simulation experience was beneficial.

Role of the tightly bound quinone for the oxygen reaction of cytochrome bo3 oxidase from Escherichia coli.

The coupling of the reaction of a tightly bound ubiquinone with the reduction of O2 in cytochrome bo3 of Escherichia coli was investigated. In the absence of the quinone, a strongly diminished rate of electrocatalytic reduction of oxygen is detected, which can be restored by adding quinones. The correlation of previous EPR data with the electrocatalytic study on mutations in the binding site at positions, Q101, D75, F93, H98, I102 and R71 reveal that the stabilization of the radical is not necessary for the oxygen reaction. The Q101 and F93 variants exhibit both well-defined catalytic i-V curves, whereas D75H, H98F, I102W and R71H exhibit broad i-V curves with large hysteresis pointing toward a strong alteration in their catalytic activity.

The CO Photodissociation and Recombination Dynamics of the W172Y/F282T Ligand Channel Mutant of Rhodobacter sphaeroides aa3 Cytochrome c Oxidase.

In the ligand channel of the cytochrome c oxidase from Rhodobacter sphaeroides (Rs aa3 ) W172 and F282 have been proposed to generate a constriction that may slow ligand access to and from the active site. To explore this issue, the tryptophan and phenylalanine residues in Rs aa3 were mutated to the less bulky tyrosine and threonine residues, respectively, which occupy these sites in Thermus thermophilus (Tt) ba3 cytochrome oxidase. The CO photolysis and recombination dynamics of the reduced wild-type Rs aa3 and the W172Y/F282T mutant were investigated using time-resolved optical absorption spectroscopy. The spectral changes associated with the multiple processes are attributed to different conformers. The major CO recombination process (44 µs) in the W172Y/F282T mutant is ~500 times faster than the predominant CO recombination process in the wild-type enzyme (~23 ms). Classical dynamic simulations of the wild-type enzyme and double mutant showed significant structural changes at the active site in the mutant, including movement of the heme a3 ring-D propionate toward CuB and reduced binuclear center cavity volume. These structural changes effectively close the ligand exit pathway from the binuclear center, providing a basis for the faster CO recombination in the double mutant.

Escherichia coli auxotroph host strains for amino acid-selective isotope labeling of recombinant proteins.

Enrichment of proteins with isotopes such as (2)H, (15)N, and (13)C is commonly carried out in magnetic resonance and vibrational spectroscopic characterization of protein structures, mechanisms, and dynamics. Although uniform isotopic labeling of proteins is straightforward, efficient labeling of proteins with only a selected set of amino acid types is often challenging. A number of approaches have been described in the literature for amino acid-selective isotope labeling of proteins, each with its own limitations. Since Escherichia coli represents the most cost-effective and widely used host for heterologous production of foreign proteins, an efficient method to express proteins selectively labeled with isotopes would be highly valuable for these studies. However, an obvious drawback is misincorporation and dilution of input isotope labels to unwanted amino acid types due to metabolic scrambling in vivo. To overcome this problem, we have generated E. coli auxotroph strains that are compatible with the widely used T7 RNA polymerase overexpression systems and that minimize metabolic scrambling. We present several examples of selective amino acid isotope labeling of simple and complex proteins with bound cofactors, as an initial guide for practical applications of these E. coli strains.

Utility of blood cultures in febrile children with UTI.

The objective of the study was to define the prevalence of bacteremia in febrile children <18 years of age diagnosed to have acute urinary tract infection (UTI). Retrospective chart review of patients diagnosed to have a UTI in the emergency department (ED) of an urban, tertiary care children's hospital was conducted Seven hundred forty-four children were discharged or admitted from the ED with a diagnosis of UTI during the study period. Thirty-six (4.8%) patient records were unavailable for review; 343 met inclusion criteria. Two hundred forty-nine patients (72.8%) had a history of fever. Blood cultures were performed on 183 (53.4%) patients. Of febrile patients, 178 (71.5%) had a blood culture performed. Seventeen of 183 (9.3%) blood cultures were positive. All (17/178, 9.5%) positive blood cultures were obtained from febrile patients. Seven of the positive blood cultures were considered to be contaminated. The prevalence of true bacteremia in febrile patients was 5.6%. All 10 patients with a true pathogen recovered from the blood culture had the same organism in their urine culture. The prevalence of bacteremia in patients younger than 2 months was 22.7% and in patients between the ages of 2 months and 36 months, 3.0%. Patients with a positive blood culture were more likely to be younger, to have been hospitalized and to have had a longer duration of hospitalization. No difference was found between patients with a positive blood culture and those without in regards to the number of days of illness before presentation, time to defervescence and mean white blood cell count. Bacteremia in children with UTI is most common in very early infancy. Children with UTI between the ages of 2 months and 12 years appear to have a low risk of bacteremia. Children who are bacteremic are likely to have identical organisms with identical antimicrobial sensitivities in both the urine and blood culture.