Dental Bite-Sized Bits: A Module for Teaching Common Oral Health Conditions to Multidisciplinary Students.

The Surgeon General's report in the year 2000 highlighted the association between chronic diseases and oral health infections. Current healthcare education programs, regrettably, report only 1 to 3 h of oral health instruction within curricula. In the years 2020-2022, as part of their respective oral health curricula, 278 first-year physician assistant and 12 pre-clinical second-year pharmacy students were invited to participate in a voluntary survey examining the effectiveness of animated succinct, online video-based oral health units. Among all student responses for the post-use survey, respondents "strongly agreed" or "agreed" that learning objectives of the unit(s) were achieved after reviewing the videos. Of the participants, 97% "strongly agreed" or "agreed" that the videos helped them understand information of which they had no prior knowledge. Similarly, 98% "strongly agreed" or "agreed" the information was appropriate for their level of knowledge. Most students, 93%, "strongly agreed" or "agreed" the exercise was a valuable learning experience. Regarding the importance of future interprofessional collaboration pertaining to a mutual patient's oral health, 95% of participants "strongly agreed" or "agreed" that they would be likely to collaborate. This study demonstrates the importance of oral health as a critical area of focus in healthcare education. The study also confirms the hypothesis that Dental Bite-Sized Bits units deliver engaging, valuable oral health education for preclinical healthcare learners, incorporating interprofessional perspectives from the disciplines of dental, pharmacy, and physician assistant.

A community of practice in dental education: A phenomenon of newcomers becoming oldtimers.

OBJECTIVES: Community of practice (CoP) members develop cooperative learning history with shared cases, techniques, and concepts. A 2020 study was designed to explore participants' perceptions toward learning in the dental education CoP. METHODS: The Institutional Review Board exempted (AZ #1355) study involved an incidental population of third- and fourth-year dental students (N = 285) and resulted in a 43.5% response rate. The online Community of Practice Assessment Scale, consisted of Likert-style, check box items, and one open-ended question. Survey responses were categorized as Strongly Agree (1), Agree (2), No opinion (3), Disagree (4), and Strongly Disagree (5). Univariate analyses and descriptive statistics were used to analyze study variables (domain, community, and practice). RESULTS: Overall the learning domain is most strongly perceived by participants with mean scores ranging from 1.59 to 1.61. Responses assessing practices within the CoP had mean scores ranging from 1.72 to 1.90. Similarly, responses assessing the community ranged from 1.65 to 1.81. "Builds Knowledge and Shared Learning" was the characteristic participants most strongly agreed as beneficial with a 1.58 mean score. Participants agree that the CoP "Captures and Stores Tacit and Explicit Knowledge" with a mean score of 1.90. There was a 25.6% response rate to the open-ended question. Two themes evolved: the need for calibration and more shared learning. CONCLUSION: Based on study results, participants strongly agreed or agreed in opinions about CoP learning resources (faculty, staff, technology, and other students) benefitting their learning. The CoP provides an optimal environment for preparing competent new dental professionals.

Power of Peers: Students' Perceptions of Pairing in Clinical Dental Education.

Prior studies have identified many benefits of peer mentoring in higher education, but the subject has not been widely examined in dental clinical education. Students at Midwestern University College of Dental Medicine-Arizona are paired with a partner for the duration of the clinical phase of education. The initial vision behind pairing was to train students in a realistic four-handed, efficient general practice model. The aim of this study was to assess the students' perceptions of the peer mentoring component of pairing third- and fourth-year dental students in the clinical phase of their education. A survey was developed to seek answers to three questions: 1) Did the students perceive that the peer mentoring supported principles of adult learning? 2) Did the students feel they were prepared to enter into the peer learning relationship? 3) What were the students' perceptions of peer mentoring in their clinical education? Data were collected through an online survey delivered to third- (n=114) and fourth-year (n=104) students at the completion of the 2014-15 school year. The 19-question survey had a 61% response rate. The benefits of pairing were found to go far beyond the initial vision of promoting a general practice model. The majority (70.1%) of responding students perceived that it added to the educational experience, and 68.5% frequently/always agreed that the mentor-mentee relationship motivated them to learn. Although the students expressed many benefits of pairing, 29.3% identified a need for more focused training prior to entering into the mentor-mentee relationship.

Redox-dependent conformational changes in cytochrome C oxidase suggest a gating mechanism for proton uptake.

A role for conformational change in the coupling mechanism of cytochrome c oxidase is the subject of controversy. Relatively small conformational changes have been reported in comparisons of reduced and oxidized crystal structures of bovine oxidase but none in bacterial oxidases. Comparing the X-ray crystal structures of the reduced (at 2.15 A resolution) and oxidized forms of cytochrome c oxidase from Rhodobacter sphaeroides, we observe a displacement of heme a(3) involving both the porphyrin ring and the hydroxyl farnesyl tail, accompanied by protein movements in nearby regions, including the mid part of helix VIII of subunit I which harbors key residues of the K proton uptake path, K362 and T359. The conformational changes in the reduced form are reversible upon reoxidation. They result in an opening of the top of the K pathway and more ordered waters being resolved in that region, suggesting an access path for protons into the active site. In all high-resolution structures of oxidized R. sphaeroides cytochrome c oxidase, a water molecule is observed in the hydrophobic region above the top of the D path, strategically positioned to facilitate the connection of residue E286 of subunit I to the active site or to the proton pumping exit path. In the reduced and reduced plus cyanide structures, this water molecule disappears, implying disruption of proton conduction from the D path under conditions when the K path is open, thus providing a mechanism for alternating access to the active site.

Proton-dependent electron transfer from CuA to heme a and altered EPR spectra in mutants close to heme a of cytochrome oxidase.

Eukaryotic cytochrome c oxidase (CcO) and homologous prokaryotic forms of Rhodobacter and Paraccocus differ in the EPR spectrum of heme a. It was noted that a histidine ligand of heme a (H102) is hydrogen bonded to serine in Rhodobacter (S44) and Paraccocus CcOs, in contrast to glycine in the bovine enzyme. Mutation of S44 to glycine shifts the heme a EPR signal from g(z) = 2.82 to 2.86, closer to bovine heme a at 3.03, without modifying other properties. Mutation to aspartate, however, results in an oppositely shifted and split heme a EPR signal of g(z) = 2.72/2.78, accompanied by lower activity and drastically inhibited intrinsic electron transfer from CuA to heme a. This intrinsic rate is biphasic; the proportion that is slow is pH dependent, as is the relative intensity of the two EPR signal components. At pH 8, the heme a EPR signal at 2.72 is most intense, and the electron transfer rate (CuA to heme a) is 10-130 s(-1), compared to wild-type at 90,000 s(-1). At pH 5.5, the signal at 2.78 is intensified, and a biphasic rate is observed, 50% fast (approximately wild type) and 50% slow (90 s(-1)). The data support the prediction that the hydrogen-bonding partner of the histidine ligand of heme a is one determinant of the EPR spectral difference between bovine and bacterial CcO. We further demonstrate that the heme a redox potential can be dramatically altered by a nearby carboxyl, whose protonation leads to a proton-coupled electron transfer process.

A conserved steroid binding site in cytochrome C oxidase.

Micromolar concentrations of the bile salt deoxycholate are shown to rescue the activity of an inactive mutant, E101A, in the K proton pathway of Rhodobacter sphaeroides cytochrome c oxidase. A crystal structure of the wild-type enzyme reveals, as predicted, deoxycholate bound with its carboxyl group at the entrance of the K path. Since cholate is a known potent inhibitor of bovine oxidase and is seen in a similar position in the bovine structure, the crystallographically defined, conserved steroid binding site could reveal a regulatory site for steroids or structurally related molecules that act on the essential K proton path.

Crystallographic location and mutational analysis of Zn and Cd inhibitory sites and role of lipidic carboxylates in rescuing proton path mutants in cytochrome c oxidase.

Cytochrome c oxidase (CcO) transfers protons from the inner surface of the enzyme to the buried O2 reduction site through two different pathways, termed K and D, and from the outer surface via an undefined route. These proton paths can be inhibited by metals such as zinc or cadmium, but the sites of inhibition have not been established. Anomalous difference Fourier analyses of Rhodobacter sphaeroides CcO crystals, with cadmium added, reveal metal binding sites that include the proposed initial proton donor/acceptor of the K pathway, Glu-101 of subunit II. Mutant forms of CcO that lack Glu-101II (E101A and E101A/H96A) exhibit low activity and eliminate metal binding at this site. Significant activity is restored to E101A and E101A/H96A by adding the lipophilic carboxylic compounds, arachidonic acid and cholic acid, but not by their non-carboxylic analogues. These amphipathic acids likely provide their carboxylic groups as substitute proton donors/acceptors in the absence of Glu-101II, as previously observed for arachidonic acid in mutants that alter Asp-132I of the D pathway. The activity of E101A/H96A is still inhibited by zinc, but this remaining inhibition is nearly eliminated by removal of subunit III, which is known to alter the D pathway. The results identify the Glu-101/His-96 site of subunit II as the site of metal binding that inhibits the uptake of protons into the K pathway and indicate that subunit III contributes to zinc binding and/or inhibition of the D pathway. By removing subunit III from E101A/H96A, thereby eliminating zinc inhibition of the uptake of protons from the inner surface of CcO, we confirm that an external zinc binding site is involved in inhibiting the backflow of protons to the active site.

Energy transduction: proton transfer through the respiratory complexes.

A series of metalloprotein complexes embedded in a mitochondrial or bacterial membrane utilize electron transfer reactions to pump protons across the membrane and create an electrochemical potential (DeltamuH+). Current understanding of the principles of electron-driven proton transfer is discussed, mainly with respect to the wealth of knowledge available from studies of cytochrome c oxidase. Structural, experimental, and theoretical evidence supports the model of long-distance proton transfer via hydrogen-bonded water chains in proteins as well as the basic concept that proton uptake and release in a redox-driven pump are driven by charge changes at the membrane-embedded centers. Key elements in the pumping mechanism may include bound water, carboxylates, and the heme propionates, arginines, and associated water above the hemes. There is evidence for an important role of subunit III and proton backflow, but the number and nature of gating mechanisms remain elusive, as does the mechanism of physiological control of efficiency.

An arginine to lysine mutation in the vicinity of the heme propionates affects the redox potentials of the hemes and associated electron and proton transfer in cytochrome c oxidase.

Cytochrome c oxidase pumps protons across a membrane using energy from electron transfer and reduction of oxygen to water. It is postulated that an element of the energy transduction mechanism is the movement of protons to the vicinity of the hemes upon reduction, to favor charge neutrality. Possible sites on which protons could reside, in addition to the conserved carboxylate E286, are the propionate groups of heme a and/or heme a(3). A highly conserved pair of arginines (R481 and R482) interact with these propionates through ionic and hydrogen bonds. This study shows that the conservative mutant, R481K, although as fully active as the wild type under many conditions, exhibits a significant decrease in the midpoint redox potential of heme a relative to Cu(A) (DeltaE(m)) of approximately equal 40 mV, has lowered activity under conditions of high pH or in the presence of a membrane potential, and has a slowed heme a(3) reduction with dithionite. Another mutant, D132A, which strongly inhibits proton uptake from the internal side of the membrane, has <4% of the activity of the wild type and appears to be dependent on proton uptake from the outside. A double mutation, D132A/R481K, is even more strongly inhibited ( approximately 1% of that of the wild type). The more-than-additive effect supports the concept that R481K not only lowers the midpoint potential of heme a but also limits a supply route for protons from the outside of the membrane used by the D132 mutant. The results are consistent with an important role of R481 and heme a/a(3) propionates in proton movement in a reversible exit path.

The protonation state of a heme propionate controls electron transfer in cytochrome c oxidase.

In cytochrome c oxidase (CcO), exergonic electron transfer reactions from cytochrome c to oxygen drive proton pumping across the membrane. Elucidation of the proton pumping mechanism requires identification of the molecular components involved in the proton transfer reactions and investigation of the coupling between internal electron and proton transfer reactions in CcO. While the proton-input trajectory in CcO is relatively well characterized, the components of the output pathway have not been identified in detail. In this study, we have investigated the pH dependence of electron transfer reactions that are linked to proton translocation in a structural variant of CcO in which Arg481, which interacts with the heme D-ring propionates in a proposed proton output pathway, was replaced with Lys (RK481 CcO). The results show that in RK481 CcO the midpoint potentials of hemes a and a(3) were lowered by approximately 40 and approximately 15 mV, respectively, which stabilizes the reduced state of Cu(A) during reaction of the reduced CcO with O(2). In addition, while the pH dependence of the F --> O rate in wild-type CcO is determined by the protonation state of two protonatable groups with pK(a) values of 6.3 and 9.4, only the high-pK(a) group influences this rate in RK481 CcO. The results indicate that the protonation state of the Arg481 heme a(3) D-ring propionate cluster having a pK(a) of approximately 6.3 modulates the rate of internal electron transfer and may act as an acceptor of pumped protons.

Water chain formation and possible proton pumping routes in Rhodobacter sphaeroides cytochrome c oxidase: a molecular dynamics comparison of the wild type and R481K mutant.

Cytochrome c oxidase (CcO) converts the energy from redox and oxygen chemistry to support proton translocation and create a transmembrane DeltamuH(+) used for ATP production. Molecular dynamics (MD) simulations were carried out to probe for the formation water chains capable of participating in proton translocation. Attention was focused on the region between and above the a and a(3) hemes where well-defined water chains have not been identified in crystallographic studies. An arginine (R481) (Rhodobacter sphaeroides numbering), positioned between the D-propionates of the hemes, had been mutated in vivo to lysine and showed to have altered activity consistent with an altered proton conductance [Qian, J., Mills, D. A., Geren, L., Wang, K. F., Hoganson, C. W., Schmidt, B., Hiser, C., Babcock, G. T., Durham, B., Millett, F., and Ferguson-Miller, S. (2004) Role of the conserved arginine pair in proton and electron transfer in cytochrome c oxidase, Biochemistry 43, 5748-5756; also see the accompanying paper by Mills et al.]. This mutant was created in silico, and the MD results for the mutant and wild type were compared to explore the effects on the formation of hydrogen-bonded water chains by this mutation. The simulations reveal the presence of hydrogen-bonded water chains that lead from E286 through the region above the hemes to the Mg(2+), and from E286 to the heme a(3) D-propionate and the binuclear center. The R481K mutant does not form as many, or as extensive, water chains as wild-type CcO, due to a new conformation of residues in a large loop between helices III and IV in subunit I, indicating a reduction in the level of water chain formation in the mutant. This loop appears to play a role in controlling the formation of hydrogen-bonded water chains above the hemes. The results suggest a possible gating mechanism for proton movement that includes key residues W172 and Y175 on the loop and F282 on helix VI.

Slow proton transfer through the pathways for pumped protons in cytochrome c oxidase induces suicide inactivation of the enzyme.

In the absence of subunit III the aa(3)-type cytochrome c oxidase exhibits a shortened catalytic life span (total number of turnovers) due to an increased probability of undergoing irreversible inactivation during steady-state turnover. Inactivation results from structural alteration of the heme a(3)-Cu(B) active site in subunit I [Hosler (2004) Biochim. Biophys. Acta 1655, 332-339]. The absence of subunit III also dramatically slows proton uptake to the active site via the D proton pathway, as well as inhibiting the proton backflow/exit pathway that connects the active site/proton pump with the outer surface of the oxidase complex. Here we demonstrate that these phenomena are linked: slow proton delivery to the active site through these pathways induces suicide inactivation, thus shortening the catalytic life span of the enzyme. Mutations that inhibit the D pathway, but not the K pathway, increase the probability of suicide inactivation. Strong inhibition of the D pathway allows suicide inactivation to occur even in the presence of subunit III. Arachidonic acid, which stimulates proton uptake by the D pathway, retards suicide inactivation. Steady-state turnover in the presence of DeltaPsi and DeltapH, which inhibits proton uptake from the inner surface of the protein, enhances suicide inactivation. Simultaneous inhibition of proton uptake from both sides of the protein by a double mutation affecting the D pathway and the proton backflow/exit pathway greatly shortens the catalytic life span of the oxidase even in the presence of subunit III. Thus, maintenance of rapid proton transfer through the D pathway and the backflow/exit pathway is one mechanism by which subunit III normally functions to prevent suicide inactivation of cytochrome c oxidase. The experiments suggest that increased lifetimes of the heme a(3) oxoferryl intermediates as well as the anionic form of Glu286 of the D pathway cause suicide inactivation in the active site.

Role of the conserved arginine pair in proton and electron transfer in cytochrome C oxidase.

A hydrogen-bonded network is observed above the hemes in all of the high-resolution crystal structures of cytochrome oxidases. It includes water and a pair of arginines, R481 and R482 (Rhodobacter sphaeroides numbering), that interact directly with heme a and the heme a(3) propionates. The hydrogen-bonded network provides potential pathways for proton release. The arginines, and the backbone peptide bond between them, have also been proposed to form part of a facilitated electron transfer route between Cu(A) and heme a. Our studies show that mutations of R482 (K, Q, and A) and R481 (K) retain substantial activity and are able to pump protons, but at somewhat reduced rates and stoichiometries. A slowed rate of electron transfer from cytochrome c to Cu(A) suggests a change in the orientation of cytochrome c binding in all but the R to K mutants. The mutant R482P is more perturbed in its structure and is altered in the redox potential difference between heme a and Cu(A): +18 mV for R482P and +46 mV for the wild type (heme a - Cu(A)). The electron transfer rate between Cu(A) and heme a is also altered from 93000 s(-1) in the wild type to 50 s(-1) in the oxidized R482P mutant, reminiscent of changes observed in a Cu(A)-ligand mutant, H260N. In neither case is the approximately 2000-fold change in the rate accounted for by the altered redox potentials, suggesting that both cause a major modification in the path or reorganization energy of electron transfer.

Histidine 386 and its role in cyclooxygenase and peroxidase catalysis by prostaglandin-endoperoxide H synthases.

Prostaglandin-endoperoxide H synthases (PGHSs) have a cyclooxygenase that forms prostaglandin (PG) G2 from arachidonic acid (AA) plus oxygen and a peroxidase that reduces the PGG2 to PGH2. The peroxidase activates the cyclooxygenase. This involves an initial oxidation of the peroxidase heme group by hydroperoxide, followed by oxidation of Tyr385 to a tyrosyl radical within the cyclooxygenase site. His386 of PGHS-1 is not formally part of either active site, but lies in an extended helix between Tyr385, which protrudes into the cyclooxygenase site, and His388, the proximal ligand of the peroxidase heme. When His386 was substituted with alanine in PGHS-1, the mutant retained <2.5% of the native peroxidase activity, but >20% of the native cyclooxygenase activity. However, peroxidase activity could be restored (10-30%) by treating H386A PGHS-1 with cyclooxygenase inhibitors or AA, but not with linoleic acid; in contrast, mere occupancy of the cyclooxygenase site of native PGHS-1 had no effect on peroxidase activity. Heme titrations indicated that H386A PGHS-1 binds heme less tightly than does native PGHS-1. The low peroxidase activity and decreased affinity for heme of H386A PGHS-1 imply that His386 helps optimize heme binding. Molecular dynamic simulations suggest that this is accomplished in part by a hydrogen bond between the heme D-ring propionate and the N-delta of Asn382 of the extended helix. The structure of the extended helix is, in turn, strongly supported by stable hydrogen bonding between the N-delta of His386 and the backbone carbonyl oxygens of Asn382 and Gln383. We speculate that the binding of cyclooxygenase inhibitors or AA to the cyclooxygenase site of ovine H386A PGHS-1 reopens the constriction in the cyclooxygenase site between the extended helix and a helix containing Gly526 and Ser530 and restores native-like structure to the extended helix. Being less bulky than AA, linoleic acid is apparently unable to reopen this constriction.

A role for subunit III in proton uptake into the D pathway and a possible proton exit pathway in Rhodobacter sphaeroides cytochrome c oxidase.

Protons are transferred from the inner surface of cytochrome c oxidase to the active site by the D and K pathways, as well as from the D pathway to the outer surface by a largely undefined proton exit route. Alteration of the initial proton acceptor of the D pathway, D132, to alanine has previously been shown to greatly inhibit oxidase turnover and slow proton uptake into the D pathway. Here it is shown that the removal of subunit III restores a substantial rate of O(2) reduction to D132A. Presumably an alternative proton acceptor for the D pathway becomes active in the absence of subunit III and D132. Thus, in the absence of subunit III cytochrome oxidase shows greater flexibility in terms of proton entry into the D pathway. In the presence of DeltaPsi and DeltapH, turnover of the wild-type oxidase or D132A is slower in the absence of subunit III. Comparison of the turnover rates of subunit III-depleted wild-type oxidase to those of the zinc-inhibited wild-type oxidase containing subunit III, both reconstituted into vesicles, leads to the hypothesis that the absence of subunit III inhibits the ability of the normal proton exit pathway to take up protons from the outside in the presence of DeltaPsi and DeltapH. Thus, subunit III appears to affect the transfer of protons from both the inner and outer surfaces of cytochrome oxidase, perhaps accounting for the long-observed lower efficiency of proton pumping by the subunit III-depleted oxidase.

Understanding the mechanism of proton movement linked to oxygen reduction in cytochrome c oxidase: lessons from other proteins.

Cytochrome c oxidase is a large intrinsic membrane protein designed to use the energy of electron transfer and oxygen reduction to pump protons across a membrane. The molecular mechanism of the energy conversion process is not understood. Other proteins with simpler, better resolved structures have been more completely defined and offer insight into possible mechanisms of proton transfer in cytochrome c oxidase. Important concepts that are illustrated by these model systems include the ideas of conformational change both close to and at a distance from the triggering event, and the formation of a transitory water-linked proton pathway during a catalytic cycle. Evidence for the applicability of these concepts to cytochrome c oxidase is discussed.

Influence of structure, pH and membrane potential on proton movement in cytochrome oxidase.

Cytochrome c oxidase (CcO) reconstituted into phospholipid vesicles and subject to a membrane potential, exhibits different characteristics than the free enzyme, with respect to effects of mutations, pH, inhibitors, and native structural differences between CcO from different species. The results indicate that the membrane potential influences the conformation of CcO and the direction of proton movement in the exit path. The importance of the protein structure above the hemes in proton exit, back leak and respiratory control is discussed.