Prevalence of Dysphonia in Metal Singers and the Impact of Vocal Education: A Subjective Analysis.

On average, the prevalence of subjectively indicated dysphonia in singers is higher than in the general population. When using extreme vocal techniques supraglottic structures are involved to protect the vocal folds. Most of the metal singers learn this specific singing style autodidactically. Vocal training can have a positive effect on the voice and prevent voice problems. The aim of the study was to find out the prevalence of dysphonia in metal singers and the impact this has on the profession or the vocal education. The subjects had to fill out an online questionnaire including two validated and standardized questionnaires about the speaking and singing voice: The short version of the Voice Handicap Index (VHI 12) and the Singing Voice Handicap Index (SVHI). The prevalence of dysphonia in metal singers is 12.2%. 9 out of 74 participants showed an increased (S)VHI score. 6 of these participants learned singing autodidactically. No dysphonia was found in any singer who sings full-time or studied singing. The correlation between education or profession and dysphonia was statistically not significant. More women than men studied or took singing lessons. This difference was statistically significant. The prevalence of 12.2% in our study is quite low and contrary to our assumption. We can assume, that a vocal education for the metal singing style is useful but not mandatory and the number of persons with dysphonia through metal singing is low. A healthy vocal technique does not necessarily have to be the result of voice lessons.

Acoustically Induced Vocal Training for Individuals With Impaired Hearing.

OBJECTIVES/HYPOTHESIS: Articulation, phonation, and resonance disorders in the speech of hearing-impaired-speakers reduces intelligibility. The study focusses on (1) whether nonacoustic feedback may facilitate the adjustment of the vocal tract, leading to increased vocal tract resonance, and (2) whether training with the feedback would be helpful for the subsequent formation of vowels. STUDY DESIGN: Prospective. METHODS: Seven profoundly hearing-impaired participants used acoustic sound waves in the frequency range of the first two vocal tract resonances applied in front of the open mouth at intensities above 1 Pa. They were asked to amplify the sound via adjusting the vocal tract. The sound waves corresponded to the first and second resonance frequencies of the vowels [u], [o], and [a]. The self-assessment of the participants and a software-based/auditory analysis was reported. RESULTS: The participants were able to enhance the acoustic signal by adjusting the vocal tract shape. The self-perception of the participants, the auditory voice analysis, and the acoustic analysis of vowels were consistent with each other. While the maximum sound pressure levels were constant, the mean sound pressure levels increased. Breathiness and hoarseness declined during the exercises. Resonance/harmonic-to-noise ratio increased, especially for the vowels [u], [o], [a]. Furthermore, the positively connoted feedback from the participants indicated easier sound production. CONCLUSION: Nonauditory feedback, based on acoustic waves, could be suitable for improving the formation of vowels. The findings are in accordance with a reduction of acoustic losses within the vocal tract.

Vocal Tract and Subglottal Impedance in High Performance Singing: A Case Study.

OBJECTIVES/HYPOTHESIS: The respiratory process is important in vocal training and in professional singing, the airflow is highly important. It is hypothesized that subglottal resonances are important to the singing voice in high performance singing. STUDY DESIGN: Single subject, prospective. METHOD: A professional soprano singer shaped her vocal tract to form the vowels [a], [e], [i], [o], and [u] at the pitch d4. We measured phonated vowels and the vocal tract impedance spectra with a deterministic noise supplied by an iPhone buzzer in the range of 200 to 4,000 Hz at closed glottis, during exhalation and during inhalation while maintaining the shape of the vocal tract. RESULTS: Measurements of the phonated vowels before and after the different glottal adjustments were highly reproducible. Vocal tract resonances and the ones resulting during respiration are reported. The impedance spectra show vowel dependent resonances with closed and open glottis. The formants of the vocal spectra are explained by including both, the vocal tract, and the subglottal resonances. CONCLUSION: The findings indicate that subglottal resonances influence the first formant as well as the singers's formant cluster in high-performance singing. The instrumental setup used for the impedance measurement allows a simple and lightweight procedure for a measurement of vocal tract and subglottal resonances.

Inhalative Treatment of Laryngitis Sicca in Patients with Sjögren's Syndrome-A Pilot Study.

Xerostomia and keratoconjunctivitis sicca are the main symptoms of Sjögren's syndrome. Often patients also suffer from laryngeal complaints, but there is a lack of specific treatment options. The aim of this study was to evaluate the effect of a liposomal inhalation therapy. Patients with Sjögren's syndrome were included and received a two-month period of liposomal inhalation therapy. The effect was evaluated by standardized questionnaires (patient-reported indices) and measurement of unstimulated whole salivary flow and glandular stiffness. Forty-five patients were included in this study. A comparison of baseline and therapeutic values demonstrated a significant improvement of the EULAR Sjögren's syndrome patient reported index (ESSPRI) with a baseline of 5.0 ± 2.1 and a therapeutic value of 4.1 ± 2.4 (p = 0.012). This improvement was mainly based on the item on dryness within this score. Overall, the therapy was well tolerated. In conclusion, an inhalative application of liposomes had a beneficial effect on the reported dryness in patients with Sjögren's syndrome. A first insight into the effect of inhalation therapy on laryngeal symptoms could thus be obtained and at the same time the basis was created on which case calculations can be carried out in the future.

Tick-borne encephalitis (TBE) in Switzerland: does the prolongation of vaccine booster intervals result in an increased risk of breakthroughs?

BACKGROUND: In 2006, the Swiss Federal Office of Public Health (FOPH) decided recommending a prolongation of vaccine booster intervals after the third dose for the prevention of tick-borne encephalitis (TBE) from 3 to 10 years. METHODS: To ascertain whether this amendment resulted in an increased rate of vaccine breakthroughs, we conducted a retrospective analysis of surveillance data collected 2000-19 by mandatory reporting to the Swiss FOPH. Fractions of breakthroughs [with 95% confidence intervals (CIs)]-0-3 years vs >3-10 years after the third vaccination dose-were compared across time periods and age groups. RESULTS: Among 3205 notified TBE cases, known vaccination status was reported in 2562 (79.9%), including 103 patients with ≥3 vaccine doses (4.0%). Among those, there were 39 patients who had received the last dose within 3 years and 48 patients in the >3-10 years group. During the 2010-19 period in which the new booster strategy was implemented there were 23 and 38 breakthroughs, respectively, and the annual breakthrough rate was 7.7 (95% CI 5.0-11.7) cases during the first 3 years after the last dose, and 5.4 (95% CI 3.9-7.5) cases in following 7 years. We observed no significant trend of TBE breakthroughs with increasing age. Increasing numbers of TBE and of vaccine breakthroughs over time have been associated with spreading endemicity and higher vaccination coverage in Switzerland. CONCLUSIONS: There is no indication that extended booster intervals resulted in an increased rate of breakthroughs, but there was a marked public health benefit with respect to increased acceptability of TBE immunization in the general population.

Reopening the Bavarian State Opera Safely: Hygiene Strategies and Incidence of COVID-19 in Artistic Staff During Theater Season 2020/2021.

Due to the drastically rising coronavirus disease (COVID-19) incidence since March 2020, social life was shut down across the globe, and most opera houses were closed. As a result, there are limited data on SARS-CoV-2 infections among artists. The Bavarian State Opera has been reopened in September 2020. This study aimed to identify the incidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection among employees in the Bavarian State Opera. In addition, the various hygiene strategies for the work groups within the institution are described. During the study period from September 1, 2020 to July 31, 2021, 10,061 nasopharyngeal swabs were obtained from 1,460 artistic staff members in a rolling system. During the entire study period, 61 individuals tested positive for SARS-CoV-2. None of the patients had a severe disease course. Compared to the seven-day-incidence per 100,000 German inhabitants, the estimated corresponding incidence among employees was lower at 37 weeks and higher or equal at 9 weeks. Among the infected individuals, 58.3% were symptomatic, 23.3% were presymptomatic, and 18.3% were asymptomatic. Forty-five percent of employees reported that they had been infected in their private environment, 41.7% suspected that their colleagues were the main contact, and 13.3% were unsure about the origin of their infection. Twenty-four diseased employees were ballet dancers, eight from the orchestra, seven from the administration, seven from the choir singers, six from the costume department, 10 from technical support, and one guest solo singer. In the 2020/2021 theater season, increased SARS-CoV-2 infections and large disease outbreaks were avoided at the Bavarian State Opera. Hygiene strategies, that existed since the beginning, was specifically designed for various work areas in the opera. Regular, mandatory PCR testing and follow-up of positive cases with the issuance of quarantine were performed. Using this disease management approach, artistic work at and reopening of the Bavarian State Opera was feasible with a well-controlled risk.

Energy transfer between the nicotinamide nucleotide transhydrogenase and ATP synthase of Escherichia coli.

Membrane bound nicotinamide nucleotide transhydrogenase (TH) catalyses the hydride transfer from NADH to NADP+. Under physiological conditions, this reaction is endergonic and must be energized by the pmf, coupled to transmembrane proton transport. Recent structures of transhydrogenase holoenzymes suggest new mechanistic details, how the long-distance coupling between hydride transfer in the peripheral nucleotide binding sites and the membrane-localized proton transfer occurs that now must be tested experimentally. Here, we provide protocols for the efficient expression and purification of the Escherichia coli transhydrogenase and its reconstitution into liposomes, alone or together with the Escherichia coli F1F0 ATP synthase. We show that E. coli transhydrogenase is a reversible enzyme that can also work as a NADPH-driven proton pump. In liposomes containing both enzymes, NADPH driven H+-transport by TH is sufficient to instantly fuel ATP synthesis, which adds TH to the pool of pmf generating enzymes. If the same liposomes are energized with ATP, NADPH production by TH is stimulated > sixfold both by a pH gradient or a membrane potential. The presented protocols and results reinforce the tight coupling between hydride transfer in the peripheral nucleotide binding sites and transmembrane proton transport and provide powerful tools to investigate their coupling mechanism.

Cross-Cultural Adaption and Validation of the Vocal Fatigue Index in German.

OBJECTIVE: The purpose of the study was to implement a cross-validation and adaption of the Vocal Fatigue Index (VFI) in German language. METHODS: The translation process for the German version (GV) of the VFI passed through three steps of translation before a final version was completed. Study subjects included 100 vocally healthy subjects and 101 voice-disordered subjects with various types of dysphonia. The internal consistency was determined using Cronbach's alpha (cron α) and the item-total analysis. Test-retest reliability was measured with the Pearson correlation coefficient. To assess the validity, the independent sample t test, the receiver-operating characteristic curve, the likelihood ratios, and Youden Index were used. RESULTS: The internal consistency across all three domains were good (#1: cron α = 0.945, #2: cron α = 0.904, and #3: cron α = 0.871) and no item of the GV of the VFI had to be deleted for further analysis relating to the item-total analysis. The test-retest reliability was high to very high (r = 0.86-0.93). Significant higher scores were revealed in voice-disordered subjects in comparison with vocally healthy subjects in all three domains (all Pvalues <0.01). Thresholds for the three domains of the GV of the VFI were determined at ≥15.5 (76.2% sensitivity and 90.0% specificity) for #1, ≥2.5 (71.5% sensitivity and 81.0% specificity) for #2, and ≤7.5 (50.5% sensitivity and 80.0% specificity) for #3. CONCLUSIONS: It can be considered that the VFI is a valid and reliable tool identifying vocal fatigue symptoms and its severity in the German-speaking population.

Fibrin Gel Suspended Autologous Chondrocytes as Cell-based Material for long-term Injection Laryngoplasty.

OBJECTIVES/HYPOTHESIS: Injection laryngoplasty of materials for unilateral vocal-fold paralysis has shown various results regarding the long-term stability of the injected material. We evaluated a fibrin-gel based cell suspension with autologous chondrocytes in-vitro and in-vivo as long-term-stable vocal-fold augmentation material in an animal model. STUDY DESIGN: This study compises an in vitro cell-culture part as well as an in vivo animal study with New Zealand White Rabbits. METHODS: In in-vitro experiments, auricular chondrocytes harvested from 24 New Zealand White Rabbits cadavers were cultivated in pellet cultures to evaluate cartilage formation for 4 weeks using long-term-stable fibrin gel as carrier. Injectability and injection volume for the laryngoplasty was determined in-vitro using harvested cadaveric larynxes. In-vivo 24 Rabbits were biopsied for elastic cartilage of the ear and autologous P1 cells were injected lateral of one vocal cord into the paraglottic space suspended in a long-term-stable fibrin gel. Histologic evaluation was performed after 2, 4, 12, and 24 weeks. RESULTS: During 12-week pellet culture, we found extracellular matrix formation and weight-stable cartilage of mature appearance. In-vivo, mature cartilage was found in two larynxes (n = 6) at 4 weeks, in four (n = 6) at 12 weeks, and in five (n = 6) at 24 weeks mostly located in the paraglottic space and sometimes with spurs into the vocalis muscle. Surrounding tissue was often infiltrated with inflammatory cells. Material tended to dislocate through the cricothyroid space into the extraglottic surrounding tissue. CONCLUSIONS: A cell-based approach with chondrocytes for permanent vocal-fold augmentation has not previously been reported. We have achieved the formation of structurally mature cartilage in the paraglottic space, but this is accompanied by difficulties with dislocated material, deformation of the augmentation, and inflammation. LEVEL OF EVIDENCE: N/A Laryngoscope, 131:E1624-E1632, 2021.

MRI- and CT-determined changes of dysphagia / aspiration-related structures (DARS) during and after radiotherapy.

PURPOSE: The concept of dysphagia/aspiration-related structures (DARS) was developed against the background of severe late side effects of radiotherapy (RT) for head and neck cancer (HNC). DARS can be delineated on CT scans, but with a better morphological discrimination on magnetic resonance imaging (MRI). Swallowing function was analyzed by use of patient charts and prospective investigations and questionnaires. METHOD: Seventeen HNC patients treated with intensity-modulated radiotherapy (IMRT) ± chemotherapy between 5/2012 - 8/2015 were included. Planning CT (computed tomography) scans and MRIs (magnetic resonance imaging) prior, during 40 Gray (Gy) radiotherapy and posttreatment were available and co-registered to delineate DARS. The RT dose of each DARS was calculated. Five patients were investigated posttreatment for swallowing function and assessed by means of various questionnaires for quality of life (QoL), swallowing, and voice function. RESULTS: By retrospective comparison of DARS volume, a significant change in four of eight DARS was detected over time. Three increased and one diminished. The risk of posttreatment dysphagia rose by every 1Gy above the mean dose (D mean) of RT to DARS. 7.5 was the risk factor for dysphagia in the first 6 months, reducing to 4.7 for months 6-12 posttreatment. For all five patients of the prospective part of swallowing investigations, a function disturbance was detected. These results were in contrast to the self-assessment of patients by questionnaires. There was neither a dose dependency of D mean DARS volume changes over time nor of dysphonia and no correlation between volume changes, dysphagia or dysphonia. CONCLUSION: Delineation of DARS on MRI co-registered to planning CT gave the opportunity to differentiate morphology better than by CT alone. Due to the small number of patients with complete MRI scans over time, we failed to detect a dose dependency of DARS and swallowing and voice disorder posttreatment.

Current problems and future avenues in proteoliposome research.

Membrane proteins (MPs) are the gatekeepers between different biological compartments separated by lipid bilayers. Being receptors, channels, transporters, or primary pumps, they fulfill a wide variety of cellular functions and their importance is reflected in the increasing number of drugs that target MPs. Functional studies of MPs within a native cellular context, however, is difficult due to the innate complexity of the densely packed membranes. Over the past decades, detergent-based extraction and purification of MPs and their reconstitution into lipid mimetic systems has been a very powerful tool to simplify the experimental system. In this review, we focus on proteoliposomes that have become an indispensable experimental system for enzymes with a vectorial function, including many of the here described energy transducing MPs. We first address long standing questions on the difficulty of successful reconstitution and controlled orientation of MPs into liposomes. A special emphasis is given on coreconstitution of several MPs into the same bilayer. Second, we discuss recent progress in the development of fluorescent dyes that offer sensitive detection with high temporal resolution. Finally, we briefly cover the use of giant unilamellar vesicles for the investigation of complex enzymatic cascades, a very promising experimental tool considering our increasing knowledge of the interplay of different cellular components.

Adjustment of Vocal Tract Shape via Biofeedback: Influence on Vowels.

The study assessed 30 nonprofessional singers to evaluate the effects of vocal tract shape adjustment via increased resonance toward an externally applied sinusoidal frequency of 900 Hz without phonation. The amplification of the sound wave was used as biofeedback signal and the intensity and the formant position of the basic vowels /a/, /e/, /i/, /o/, and /u/ were compared before and after a vocal tract adjustment period. After the adjustment period, the intensities for all vowels increased and the measured changes correlated with the participants' self-perception.The diferences between the second formant position of the vowels and the applied frequency influences the changes in amplitude and in formant frequencies. The most significant changes in formant frequency occurred with vowels that did not include a formant frequency of 900 Hz, while the increase in amplitude was the strongest for vowels with a formant frequency of about 900 Hz.

Laryngeal and Pharyngeal Movements During Inner Singing: A Cross-Sectional Study.

INTRODUCTION: Laryngeal and pharyngeal activity during inner singing is discussed in the context of vocal hygiene. Inner singing is defined as imagined singing, reading music silently, and listening to vocal music. When vocal rest is prescribed, doctors, speech therapists, and voice pedagogues recommend avoiding listening to music or reading music silently, since it is suggested that inner singing unconsciously influences the glottis, and thus moves the vocal folds involuntarily. The aim of this study was to compare the degree to which involuntary laryngeal and/or pharyngeal activity occur during inner singing, inner speech, and at rest, and to evaluate if current recommendations concerning vocal hygiene are still reasonable. MATERIAL AND METHOD: Thirty vocally healthy participants were examined transnasally with a flexible videoendoscope. The sample consisted of 10 nonsingers, 10 lay singers, and 10 professional singers. Participants were examined during five tasks including rest, silent reading, imagining a melody, listening to music, and reading music. Two medical doctors specializing in phoniatrics analyzed the videos both qualitatively and quantitatively. RESULTS: During the endoscopic examination, the raters identified movements at the base of the tongue, the posterior and lateral pharynx wall, the arytenoid cartilage, and the vocal folds. The inner singing tasks showed significantly more laryngeal movements as well as significantly more glottal closures than the control tasks (at rest, silent reading). Pharyngeal structures did not show an increase in activity during inner singing. These findings were independent of the level of proficiency in singing. CONCLUSION: When total vocal rest is prescribed, patients should also be advised to avoid music imagination. Still, further research is needed to survey in detail the actual effects of these involuntary movements during inner singing on the regeneration process of vocal fold healing.

[Training Curriculum for the Certificate "Diagnostics and Therapy of Oropharyngeal Dysphagia, including FEES", of the German Society for Phoniatrics and Pedaudiology and the German Society for Otolaryngology, Head and Neck Surgery]. / Ausbildungscurriculum zum Zertifikat "Diagnostik und Therapie oropharyngealer Dysphagien, inklusive FEES" der Deutschen Gesellschaft für Phoniatrie und Pädaudiologie und der Deutschen Gesellschaft für Hals-Nasen-Ohren-Heilkunde, Kopf- und Hals-Chirurgie.

BACKGROUND: In Germany, about 5 million people of all ages suffer from dysphagia. Due to demographic change and improved medical care, the incidence of swallowing disorders is expected to increase. Dysphagia is associated with an increased morbidity and mortality and leads to a considerable financial burden on the health systems. The two most common causes of dysphagia are neurological disorders and head and neck cancer. Diagnostics and therapy have developed continuously over the past decades. In particular, the flexible endoscopic evaluation of swallowing (FEES) has become an established part of dysphagia diagnostics. RESULTS: The certificate "Diagnostics and Therapy of Oropharyngeal Dysphagia, incl. FEES" was developed by the German Society for Phoniatrics and Pedaudiology (DGPP) and the German Society for Otolaryngology, Head and Neck Surgery (DGHNO KHC) in cooperation with the German Professional Association for Phoniatrics and Pedaudiology and the German Professional Association of Otolaryngologists.It consists of three parts: the modules (A, B and C), the indirect supervision and a practical examination. Structure, detailed contents and requirements for obtaining the certificate are described in the following article. The qualification of the lecturers and auditors are also defined. CONCLUSION: The systematic training serves the quality assurance and establishment of standards in the diagnostics and therapy of oropharyngeal dysphagia in the area of phoniatrics and ear, nose and throat medicine.

[Differentiation of ultrasonographic hypoechoic head and neck lesions]. / Differentialdiagnostik sonographisch echoarmer Kopf-Halsläsionen.

AIM: To evaluate ultrasonographic hypoechoic lesions (HEL) of the head and neck region to predict their entity and plan surgery. METHODS: Patients with HEL were included that were further analysed by B-mode ultrasound (US), colour-coded duplex sonography (CDS), contrast enhanced US (CEUS), and strain elastography (SE). RESULTS: 184 patients were included. Level VIII, II, and I were affected frequently with 103, 40, and 21 HEL. The cohort comprised 40 lymph node disorders, 101 salivary gland diseases, 31 cystic lesions, and 12 other rarer entities. HEL in level II were significantly larger than in level I and VIII (p < 0.001). HEL in level VI showed less vascularisation than in level VIII in CDS (p < 0.01). There were no differences in B-mode criteria, SE, or CEUS between HEL in the different neck levels. Patients with cystic lesions were significantly younger than patients with metastases or Warthin's tumours (p = 0.026, 0.028). Pleomorphic adenomas were significantly smaller than cystic lesions (p < 0.0006), lymphomas (p = 0.026), metastases (p = 0.0003), or Warthin's tumours (p = 0.034). In CDS and CEUS, cystic lesions showed significantly less vascularisation and perfusion than lymphomas (p = 0.014) and Warthin's tumours (p < 0.0001), while pleomorphic adenomas were stiffer than cystic lesions in SE (p = 0.0006). CONCLUSION: Predicting lesion's entity is still challenging. The combination of different ultrasonographic criteria helped selecting patients that needed intraoperative fresh frozen section with possible extended surgery and profited from intraoperative nerve monitoring.

The proton pumping bo oxidase from Vitreoscilla.

The cytochrome bo3 quinol oxidase from Vitreoscilla (vbo3) catalyses oxidation of ubiquinol and reduction of O2 to H2O. Data from earlier studies suggested that the free energy released in this reaction is used to pump sodium ions instead of protons across a membrane. Here, we have studied the functional properties of heterologously expressed vbo3 with a variety of methods. (i) Following oxygen consumption with a Clark-type electrode, we did not observe a measurable effect of Na+ on the oxidase activity of purified vbo3 solubilized in detergent or reconstituted in liposomes. (ii) Using fluorescent dyes, we find that vbo3 does not pump Na+ ions, but H+ across the membrane, and that H+-pumping is not influenced by the presence of Na+. (iii) Using an oxygen pulse method, it was found that 2 H+/e- are ejected from proteoliposomes, in agreement with the values found for the H+-pumping bo3 oxidase of Escherichia coli (ecbo3). This coincides with the interpretation that 1 H+/e- is pumped across the membrane and 1 H+/e- is released during quinol oxidation. (iv) When the electron transfer kinetics of vbo3 upon reaction with oxygen were followed in single turnover experiments, a similar sequence of reaction steps was observed as reported for the E. coli enzyme and none of these reactions was notably affected by the presence of Na+. Overall the data show that vbo3 is a proton pumping terminal oxidase, behaving similarly to the Escherichia coli bo3 quinol oxidase.

Adjustment of the Vocal Tract Shape via Biofeedback: A Case Study.

In this study, an adjustment of the vocal tract shape toward selected sound waves in the frequency range of the first and second formants without phonation is discussed. The sound waves of a loudspeaker in front of the open mouth and amplified by the vocal tract are used as biofeedback signals. It is shown that the resonance amplification of the vocal tract complies with the concept of forced oscillation, with the driver being the sound source and the resonator being the vocal tract. An adjustment toward increased amplification via vocal tract resonance can be related to smaller bandwidths and lower damping. Furthermore, the applied adjustment frequencies are preserved as vocal tract resonances during exhalation and even phonation. This novel form of biofeedback might enrich standard voice training procedures by exercises without phonation.

Rapid Electron Transfer within the III-IV Supercomplex in Corynebacterium glutamicum.

Complex III in C. glutamicum has an unusual di-heme cyt. c1 and it co-purifies with complex IV in a supercomplex. Here, we investigated the kinetics of electron transfer within this supercomplex and in the cyt. aa3 alone (cyt. bc1 was removed genetically). In the reaction of the reduced cyt. aa3 with O2, we identified the same sequence of events as with other A-type oxidases. However, even though this reaction is associated with proton uptake, no pH dependence was observed in the kinetics. For the cyt. bc1-cyt. aa3 supercomplex, we observed that electrons from the c-hemes were transferred to CuA with time constants 0.1-1 ms. The b-hemes were oxidized with a time constant of 6.5 ms, indicating that this electron transfer is rate-limiting for the overall quinol oxidation/O2 reduction activity (~210 e-/s). Furthermore, electron transfer from externally added cyt. c to cyt. aa3 was significantly faster upon removal of cyt. bc1 from the supercomplex, suggesting that one of the c-hemes occupies a position near CuA. In conclusion, isolation of the III-IV-supercomplex allowed us to investigate the kinetics of electron transfer from the b-hemes, via the di-heme cyt. c1 and heme a to the heme a3-CuB catalytic site of cyt. aa3.