Pulmonary thromboembolism in hospitalised patients with COVID-19: a retrospective national study of patients managed in critical care and ward environments in Scotland.

OBJECTIVES: To assess for increase in pulmonary thromboembolism (PTE) in hospitalised patients with COVID-19, in both critical care and ward environments. SETTING: We reviewed all CT pulmonary angiograms (CTPA) performed in Scotland between 23 March 2020 and 31 May 2020 and identified those with COVID-19 using either classical radiological appearances or positive COVID-19 PCR swab. PARTICIPANTS: All hospitalised patients in Scotland with COVID-19 between 23 March 2020 and 31 May 2020 who underwent a CTPA. PRIMARY OUTCOME MEASURE: To assess if the rate of PTE was increased in those with COVID-19 compared with previously published figures of hospitalised patients. SECONDARY OUTCOME MEASURES: To assess the effect of right heart strain or requirement for critical care on mortality. RESULTS: 3401 CTPAs were reviewed. 192 were positive for PTE in patients with evidence of COVID-19 either real-time PCR swab positive for SARS-CoV-2 (n=104) or having radiological changes consistent with COVID-19 (n=88). The total number of hospital admissions in Scotland between 23rd March 2020 and 31st May 2020 with COVID-19 was 5195. The incidence of PTE during this time was 3.7% in all patients admitted to all hospitals in Scotland with COVID-19 during this period. 475 hospitalised patients were managed in critical care (both level 2 and level 3 care), in whom the incidence of PTE was 6% (n=29). 4720 patients did not require admission to critical care, in whom the incidence of PTE was 3.5% (n=163). There was increased risk of death with right heart strain (25/52 vs 128/140 (p<0.01)) and in critical care (15/29 vs 146/163 (p<0.01)). CONCLUSIONS: We have demonstrated an increased risk of PTE in critical care and ward-based environments. Further studies are required to establish effective prophylactic anticoagulation in this group.

The role of per cent predicted 6-min walk distance in pulmonary arterial hypertension.

Absolute 6-min walk distance (6MWD) predicts mortality in pulmonary arterial hypertension (PAH), but varies greatly between normal individuals due to physiological factors such as age, sex, height and weight. The % predicted 6MWD adjusts for these factors and may predict mortality more reliably. The aim of the study was to compare the strength of mortality prediction by absolute and % predicted 6MWD in PAH at baseline and on treatment. % predicted 6MWD was calculated using four different reference equations in 137 PAH patients (idiopathic and connective tissue disease associated) diagnosed between November 2000 and November 2009. Cox proportional hazards and receiver-operating characteristic (ROC) analyses were used to compare the prognostic strength of absolute and % predicted 6MWD. % predicted 6MWD was predictive of all-cause mortality at baseline (hazard ratio 0.74-0.83 per 10% increase; p<0.05) and on treatment (0.67-0.75 per 10% increase; p<0.01), but each respective area under the ROC curve was not different from that of absolute 6MWD for predicting 2-yr mortality at baseline (absolute versus % predicted: 0.74 versus 0.71-0.75) or on treatment (0.77 versus 0.72-0.78). In conclusion, % predicted 6MWD may help clinicians interpret the 6-min walk test, but its prognostic value is not superior to that of absolute 6MWD.

Pearls and Pitfalls in MR Enterography Interpretation for Pediatric Patients.

Magnetic resonance (MR) enterography is now the preferred modality for evaluation of acute and chronic presentations of Crohn disease in pediatric patients. There has been increasing standardization in the performance and interpretation of these studies, given the growth in volume and impact on clinical management. This article will focus on technical considerations in the performance of MR enterography in children and adolescents, as well as "do not miss" findings on MR enterography that will impact clinical management and potential problems encountered with MR enterography that may limit its diagnostic utility in some patients.

Identification of novel candidate genes associated with cleft lip and palate using array comparative genomic hybridisation.

AIM AND METHOD: We analysed DNA samples isolated from individuals born with cleft lip and cleft palate to identify deletions and duplications of candidate gene loci using array comparative genomic hybridisation (array-CGH). RESULTS: Of 83 syndromic cases analysed we identified one subject with a previously unknown 2.7 Mb deletion at 22q11.21 coinciding with the DiGeorge syndrome region. Eighteen of the syndromic cases had clinical features of Van der Woude syndrome and deletions were identified in five of these, all of which encompassed the interferon regulatory factor 6 (IRF6) gene. In a series of 104 non-syndromic cases we found one subject with a 3.2 Mb deletion at chromosome 6q25.1-25.2 and another with a 2.2 Mb deletion at 10q26.11-26.13. Analyses of parental DNA demonstrated that the two deletion cases at 22q11.21 and 6q25.1-25.2 were de novo, while the deletion of 10q26.11-26.13 was inherited from the mother, who also has a cleft lip. These deletions appear likely to be causally associated with the phenotypes of the subjects. Estrogen receptor 1 (ESR1) and fibroblast growth factor receptor 2 (FGFR2) genes from the 6q25.1-25.2 and 10q26.11-26.13, respectively, were identified as likely causative genes using a gene prioritization software. CONCLUSION: We have shown that array-CGH analysis of DNA samples derived from cleft lip and palate subjects is an efficient and productive method for identifying candidate chromosomal loci and genes, complementing traditional genetic mapping strategies.

Regulation of iNOS by the p44/42 mitogen-activated protein kinase pathway in human melanoma.

Activating mutations of the genes for NRAS and BRAF, components of the p44/42 mitogen-activated protein kinase (MAPK) pathway, are common findings in melanoma. Recent evidence in several nonmelanoma cell systems supports the regulation of the inducible nitric oxide synthase (iNOS) gene by this pathway. On the basis of our data showing that melanoma iNOS expression predicts shortened patient survival, we formulated the hypothesis that activating mutations of NRAS or BRAF, which lead to constitutive activation of the p44/42 MAPK pathway, drive iNOS expression in human melanoma. In the present study, we have shown that inhibition of melanoma iNOS activity by S-methylisothiourea leads to decreased cell proliferation, confirming the importance of iNOS activity for melanoma cell growth. Regulation of melanoma iNOS expression by the p44/42 MAPK pathway was demonstrated by inhibition of the pathway by U0126, and by BRAF RNA interference. To explore this regulatory pathway in human tissue, 20 melanoma tumors were examined for NRAS and BRAF mutations, immunohistochemical evidence of ERK phosphorylation, and iNOS expression. A significant association was found among these three features. We conclude that in human melanoma, activating mutations of NRAS and BRAF drive constitutive iNOS expression and, implicitly, nitric oxide production, contributing to the poor survival of these patients.

Diagnosis of prediabetes in cats: glucose concentration cut points for impaired fasting glucose and impaired glucose tolerance.

Diabetes is typically diagnosed in cats once clinical signs are evident. Diagnostic criteria for prediabetes in cats have not been defined. The objective of the study was to establish methodology and cut points for fasting and 2-h blood glucose concentrations in healthy client-owned senior cats (≥8 yr) using ear/paw samples and a portable glucose meter calibrated for feline blood. Of the 78 cats, 27 were ideal (body condition score [BCS] 4 or 5 of 9), 31 overweight (BCS 6 or 7), and 20 obese (BCS 8 or 9); 19 were Burmese and 59 non-Burmese. After an 18-24-h fast and an ear/paw blood glucose measurement using a portable glucose meter, glucose (0.5 g/kg bodyweight) was administered intravenous and blood glucose measured at 2 min and 2 h. Cut points for fasting and 2-h glucose concentrations were defined as the upper limits of 95% reference intervals using cats with BCS 4 or 5. The upper cut point for fasting glucose was 6.5 mmol/L. Of the overweight and obese cats, 1 (BCS 7) was above this cut point indicating evidence of impaired fasting glucose. The cut point for 2-h glucose was 9.8 mmol/L. A total of 7 cats (4 with BCS 8 or 9 including 1 Burmese; 3 with BCS 6 or 7, non-Burmese) were above this cut point and thus had evidence of impaired glucose tolerance. In conclusion, the methodology and cutpoints for diagnosis of prediabetes are defined for use in healthy cats 8 yr and older with a range of BCSs.

Dosing obese cats based on body weight spuriously affects some measures of glucose tolerance.

The primary objective was to investigate whether dosing glucose by body weight results in spurious effects on measures of glucose tolerance in obese cats because volume of distribution does not increase linearly with body weight. Healthy research cats (n = 16; 6 castrated males, 10 spayed females) were used. A retrospective study was performed using glucose concentration data from glucose tolerance and insulin sensitivity tests before and after cats were fed ad libitum for 9 to 12 mo to promote weight gain. The higher dose of glucose (0.5 vs 0.3 g/kg body weight) in the glucose tolerance tests increased 2-min glucose concentrations (P < 0.001), and there was a positive correlation between 2-min and 2-h glucose (r = 0.65, P = 0.006). Two-min (P = 0.016 and 0.019, respectively), and 2-h (P = 0.057 and 0.003, respectively) glucose concentrations, and glucose half-life (T1/2; P = 0.034 and <0.001 respectively) were positively associated with body weight and body condition score. Glucose dose should be decreased by 0.05 g for every kg above ideal body weight. Alternatively, for every unit of body condition score above 5 on a 9-point scale, observed 2-h glucose concentration should be adjusted down by 0.1 mmol/L. Dosing glucose based on body weight spuriously increases glucose concentrations at 2 h in obese cats and could lead to cats being incorrectly classified as having impaired glucose tolerance. This has important implications for clinical studies assessing the effect of interventions on glucose tolerance when lean and obese cats are compared.

Left anterior prefrontal activation increases with demands to recall specific perceptual information.

Results from neuroimaging studies have led to competing theories regarding the contributions of prefrontal regions to memory formation and retrieval. To investigate this issue, we used event-related functional magnetic resonance imaging to assess prefrontal activation during encoding and retrieval of pictures of objects. Responses to studied and unstudied objects at retrieval were compared between two tests with differing demands for the specificity of information to be retrieved (source vs old-new recognition). Results showed that bilateral ventral [Brodmann's areas (BA) 44, 45, and 47] and right dorsal (BA 9) prefrontal regions were activated during both encoding and retrieval, but activity in these regions was not reliably modulated by the specificity of information to be retrieved. A region in left anterior prefrontal cortex (BA 10/46) was reliably activated during retrieval trials, and activation in this region increased with demands to retrieve perceptually detailed information about studied objects. Our results show that left anterior prefrontal cortex is engaged during the monitoring and evaluation of specific memory characteristics at retrieval-a process critical for accurate episodic remembering.

The electronic and magnetic properties of rubredoxin: a low-temperature magnetic circular dichroism study.

Oxidized rubredoxin from Clostridium pasteurianum has been investigated by magnetic circular dichroism (MCD) spectroscopy over the temperature range 1.5 to 150 K and at magnetic fields between 0 and 4.5 tesla. The results show that studies of the temperature and field dependence of MCD transitions afford insight into the polarization of electronic transitions for ground states with large g-value anisotropy, in addition to estimates of ground-state g values and zero-field splitting parameters. In agreement with the assignment made by Eaton and Lovenberg (Eaton, W.A. and Lovenberg, W. (1973) in Iron-Sulfur Proteins, Vol. II (Lovenberg, W., ed.), pp. 131-162, Academic Press, New York), the ultraviolet-visible spectrum of oxidized rubredoxin is assigned to two S----Fe(III) charge transfer transitions (both 6A1----6T2 under tetrahedral symmetry), each spanning a range of 650-430 nm and 430-330 nm, respectively. The observed splitting in each of these transitions is attributed to a predominant axial distortion in the excited state resulting in effective D2d symmetry.

Interaction of some trialkyl phosphorothiolates with acetylcholinesterase. Characterization of inhibition, aging and reactivation.

The reaction of bovine erythrocyte acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) with a set of structurally related phosphorothiolates was studied in order to investigate the properties of the phosphorylated enzymes and to identify the leaving group. OOS- and OOS-trimethyl phosphorothiolates and their triethyl analogues inhibit acetylcholinesterase reversibly and by progressive inhibition, and the phosphorylated enzymes undergo both spontaneous reactivation and aging. For each compound the enzyme-inhibitor dissociation constant, and the rate constants for inhibition (ka), reactivation and aging have been derived. The OOS-compounds are more potent inhibitors than the OOS-compounds, and the derived inhibited enzymes reactivate and age faster. By comparing reactivation and aging rate constants with those obtained from phosphorylated enzymes of known structure it was concluded that the leaving group of during phosphorylation is the S-alkyl. SSS-trimethyl and -triethyl phosphorothiolates also form reversible complexes and inhibit the enzyme progressively. With these inhibitors the phosphorylated enzymes did not reactivate either spontaneously or in response to oximes under conditions successful for the other inhibitors. The ka values (37 degrees C, pH 7.4) range from 30 M-1 X min-1 (OOS-trimethyl phosphorothiolate) to 6.7 X 10(3) M-1 X min-1 (OOS-triethyl phosphorothiolate) as compared to 1.25 X 10(5) M-1 X min-1 determined for isomalathion (O, S-dimethyl S-(1,2-dicarbethoxyethyl)-phoshporodithioate), which was used as one of the reference compounds. If the inhibitory potency of the trialkyl phosphorothiolates is calculated from measurements made after a fixed preincubation time the results in ka values will be misleading.

Variable temperature magnetic circular dichroism studies of reduced nitrogenase iron proteins and [4Fe-4S]+ synthetic analog clusters.

Variable temperature magnetic circular dichroism (VTMCD) and EPR spectroscopies have been used to investigate the ground and excited-state properties of [4Fe-4S]+ clusters in Mo- and V-nitrogenase Fe-proteins from Azotobacter vinelandii and two synthetic analog clusters, [Fe4S4(SEt)4]3- and [Fe4S4(SC6H11)4]3-. The results indicate similar [4Fe-4S]+ clusters with analogous S = 1/2 and S = 3/2 ground states in both Fe-proteins. However, the Fe-proteins do differ in terms of the medium effects on the S = 1/2 and S = 3/2 spin mixtures in frozen solution. By utilizing medium effects in both Fe-proteins, the VTMCD characteristics of both the S = 1/2 and S = 3/2 forms of the [4Fe-4S]+ have been determined. Together with the VTMCD studies of [Fe4S4(SEt)4]3- and [Fe4S4(SC6H11)4]3-, which are shown to be predominantly S = 1/2 and 3/2, respectively, in frozen DMF/toluene solutions, the results demonstrate that the form of the VTMCD spectra provides a means of identifying and distinguishing S = 1/2 and S = 3/2 [4Fe-4S]+ clusters. Ground state zero-field splitting parameters for the S = 3/2 clusters are determined for both Fe-proteins. In addition to spin state heterogeneity, samples of the Mo-nitrogenase Fe-protein in the presence of 50% (v/v) ethylene glycol were found to exhibit heterogeneity in the S = 1/2 resonance. A rapidly relaxing axial resonance, g perpendicular = 1.94 and g parallel = 1.82, was observed in addition to the characteristic rhombic resonance, g = 2.05, 1.94 and 1.87. The origin of the heterogeneity exhibited by [4Fe-4S]+ clusters in frozen solution is discussed in light of these results.

Spectroscopic characterization of the alternate form of S-methylcoenzyme M reductase from Methanobacterium thermoautotrophicum (strain delta H).

Two forms (MR1 and MR2) of S-methylcoenzyme M reductase were purified from Methanobacterium thermoautotrophicum (strain delta H) as recently described (Rospert, S., Linder, D., Ellerman, J. and Thauer, R.K. (1990) Eur. J. Biochem. 194, 871-877). MR2 was at least 50-fold more active than MR1, independent of assay conditions. The two forms are spectroscopically similar, but not identical, by UV-visible, magnetic circular dichroism and resonance Raman spectroscopies. MR2 exhibited an EPR signal corresponding to 20% of the enzyme-bound nickel. Strong EPR signals similar to those previously assigned to Ni(I)F430 bound to methylreductase in Methanobacterium thermoautotrophicum (strain Marburg) (Albracht, S.P.J., Ankel-Fuchs, D., Bocher, R., Ellerman, J., Moll, J., Van der Zwann, J.W. and Thauer, R.K. (1988) Biochim. Biophys. Acta 955, 86-102) were observed in MR2-rich, log-phase, as well as in MR1-rich, slow-growing bacteria. Log-phase cells had dramatically different EPR spectra depending on whether they were removed from the fermenter (under gas flow) before or after cooling to 10 degrees C. EPR spectra of slow-growing cells were insensitive to harvesting conditions. The possible biological significance of the alternate form of methylreductase is discussed.

Spectroscopic studies of the seven-iron-containing ferredoxins from Azotobacter vinelandii and Thermus thermophilus.

The seven-iron-containing ferredoxins from Azotobacter vinelandii and Thermus thermophilus have been investigated by low-temperature magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectroscopies and room temperature ultraviolet-visible absorption spectroscopy. The results confirm the presence of one trinuclear and one tetranuclear iron-sulfur cluster in both ferredoxins and facilitate comparison of the electronic and magnetic properties of the oxidized and reduced [3Fe-xS] clusters. MCD magnetization data are consistent with an S = 2 ground state for both reduced [3Fe-xS] clusters, but indicate differences in the rhombicity of the zero-field splittings. The data permit rationalization of the absence of a delta M = 4 EPR transition for the reduced [3Fe-xS] cluster in A. vinelandii ferredoxin I. Spectroscopic studies of anaerobically isolated A. vinelandii ferredoxin I do not support the hypothesis that the [3Fe-xS] cluster arises as a result of aerial oxidative damage to a [4Fe-4S] cluster during isolation. The possibility that two distinct forms of [3Fe-xS] clusters can exist in A. vinelandii ferredoxin I was investigated by spectroscopic studies as a function of pH. The results reveal two distinct and interconvertible forms of the reduced [3Fe-xS] cluster, but do not permit rationalization of the inconsistencies in the structural data that have been reported for the oxidized clusters.

Quarter field resonance and integer-spin/half-spin interaction in the EPR of Thermus thermophilus ferredoxin. Possible new fingerprints for three iron clusters.

We describe two new characteristics of the EPR of the seven-iron containing ferredoxin from Thermus thermophilus. First, the reduced state of the 3Fe center, which has traditionally been considered to be EPR-silent, has been found to exhibit a delta m = 4 transition, which is unique for Fe-S centers. This signal is similar to that of high-spin Fe2+-EDTA and supports the suggestion that the ground electronic state of the 3Fe cluster is S = 2. Second, we have recorded the EPR spectrum of the fully reduced protein at 9 and 15 GHz and found that changes occur in the signal which are consistent with a weak electronic spin-spin interaction between the [4Fe-4S]+ (S = 1/2) and the reduced 3Fe center. A theoretical explanation is given for the observation of interaction signals with constant effective g values.

Oxidation-reduction properties of the ferredoxin-linked glutamate synthase from spinach leaf.

Oxidation-reduction titrations have been conducted to determine the midpoint potential (Em) values of the three electron-carrying prosthetic groups of the ferredoxin-linked glutamate synthase isolated from spinach leaves. Titrations using electron paramagnetic resonance (EPR) signals to monitor the oxidation state of the [3Fe-4S]+,0 cluster found in the enzyme, indicated the presence of a single n = 1 component with Em = -170 mV at pH 7.7. Titrations using absorbance changes in the visible region to monitor the oxidation states of the FAD and FMN groups present in the enzyme could be fit to a single n = 2 Nernst curve with Em = -180 mV at pH 7.7. The magnitude of the absorbance change observed during this titration accounts for all of the FMN and FAD found in the enzyme, indicating that the two flavins are either isopotential or differ in Em by less than about 30 mV. Neither optical nor EPR titrations gave any evidence for the presence of stable flavin free radicals. These results represent the first characterization of the redox properties of the prosthetic groups of a ferredoxin-dependent glutamate synthase.

Low-temperature magnetic circular dichroism spectra and magnetisation curves of 4Fe clusters in iron-sulphur proteins from Chromatium and Clostridium pasteurianum.

The magnetic circular dichroism (MCD) spectra of the 4Fe clusters in the iron-sulphur proteins high-potential iron protein from Chromatium and the 8Fe ferredoxin from Clostridium pasteurianum have been measured over the wavelength range 300-800 nm at temperatures between approx. 1.5 and 50 K and at magnetic fields up to 5 tesla. In both cases the proteins have been studied in the oxidized and reduced states. The reduced state of high-potential iron protein gives a temperature-independent MCD spectrum up to 20 K, confirming the diamagetism of this state at low temperature. The MCD spectrum of samples of oxidized ferredoxin invariably show the presence of a low concentration of a paramagnetic species, in agreement with the observation that the EPR spectrum always shows a signal at g = 2.01. The paramagnetic MCD spectrum runs across the whole of the wavelength range studied and therefore most probably originates from an iron-sulphur centre. The diamagnetic component of the MCD spectrum of oxidized ferredoxin is very similar to that of reduced high-potential iron protein. The low-temperature MCD spectra of oxidized high-potential iron protein and reduced ferredoxin reveal intense, temperature-dependent bands. The spectra are highly structured with that of high-potential iron protein showing a large number of electronic transitions across the visible region. The MCD spectra of the two different oxidation levels are quite distinctive and should provide a means of establishing the identity of these state of 4Fe clusters in more complex proteins. MCD magnetisation curves have been constructed from detailed studies of the field and temperature dependence of the MCD spectra of the two paramagnetic oxidation states. These plots can be satisfactorily fitted to the theoretically computed curves for an S = 1/2 ground state with the g factors experimentally determined by EPR spectroscopy. The low-temperature MCD spectra of the reduced 2Fe-2S ferredoxin from Spirulina maxima are also presented and MCD magnetisation curves plotted and fitted to the experimentally determined g factors.

Spectroscopic characterization of flavocytochrome c-552 from the photosynthetic purple sulfur bacterium Chromatium vinosum.

The identities of the axial ligands to the two hemes of the flavocytochrome c-552 isolated from the photosynthetic purple sulfur bacterium Chromatium vinosum have been investigated by visible/near-infrared absorption and magnetic circular dichroism (MCD) spectroscopies, with parallel electron paramagnetic resonance (EPR) studies. One of the hemes has histidine and methionine as axial ligands and has a local environment that is relatively insensitive to the composition of the bulk medium. The second heme, the local environment of which is sensitive to changes in the composition of the bulk medium, exists as a mixture of two forms, only one of which has histidine/methionine axial ligation. On the basis of its EPR characteristics, the other form most likely has histidine/lysine axial ligation. In aqueous solution near neutral pH, more than half of the second heme is present as the histidine/lysine form, while in 50:50 water/ethylene glycol the histidine/methionine form is the dominant one.

The three-iron cluster in a ferredoxin from Desulphovibrio gigas. A low-temperature magnetic circular dichroism study.

Ferredoxin II from Desulphovibrio gigas is a tetrameric protein containing a novel iron-sulphur cluster consisting of three iron atoms. The low-temperature magnetic circular dichroism (MCD) spectra of the oxidized and dithionite-reduced forms of ferredoxin II have been measured over the wavelength range approx. 300-800 nm. Both oxidation levels of the cluster are shown to be paramagnetic, although only the oxidized form gives an EPR signal. MCD magnetization curves have been constructed over the temperature range approx. 1.5-150 K and at fields between 0 and 5.1 Tesla. The curve for the oxidized protein can be fitted to a ground state of spin S = 1/2 with an isotropic g factor of 2.01. There is evidence for the thermal population of a low-lying electronic state above 50 K. The reduced protein gives a distinctive set of magnetization curves that are tentatively assigned to a ground state of S = 2, with a predominantly axial zero-field distortion that leaves the doublet Ms = +/-2 lowest in energy. The zero-field components have a maximum energy spread of approx. 15 cm-1. which places an upper limit of 4 cm-1 on the axial zero-field parameter D. The MCD spectra of the oxidized and reduced forms of the cluster are quite distinctive from one another. The spectra of the oxidized state are also different from those of oxidized high-potential iron protein from Chromatium and should provide a useful criterion for distinguishing between four- and three-iron clusters in their highest oxidation levels.

Axial heme ligation in the cytochrome bc1 complexes of mitochondrial and photosynthetic membranes. A near-infrared magnetic circular dichroism and electron paramagnetic resonance study.

The combination of EPR and low-temperature near-IR magnetic circular dichroism spectroscopies have been used to investigate the axial ligation of the cytochromes in the cytochrome bc1 complexes from bovine heart mitochondria, Rhodobacter capsulatus, Rhodobacter sphaeroides, and Rhodospirillum rubrum, and the purified cytochromes c1 from bovine heart mitochondria, Rb. capsulatus and Rb. sphaeroides. The possibility of axial ligation of cytochrome c1 by the amino terminus of the polypeptide was also assessed by acetylating the N-terminus of Rb. capsulatus cytochrome c1 and comparing the properties of the acetylated and unmodified samples. The results are consistent with bis-histidine axial ligation for the high- and low-potential b-type cytochromes and histidine/methionine axial ligation for the c1-type cytochrome in the intact cytochrome bc1 complexes. Purified samples of cytochrome c1 are mixtures of two forms, one with histidine/methionine and the other with bis-histidine axial ligation. The form with bis-histidine axial ligation is also assembled in the M183L mutant of the Rb. capsulatus cyt bc1 complex in which the methionine residue coordinating cyt c1 is replaced by a leucine. The bis-histidine form appears to be an artifact of dissociation of cytochrome c1 from the cytochrome bc1 complex and is greatly enhanced particularly in the bacterial cytochromes c1 by sample handling and the addition of 50% (v/v) ethylene glycol or glycerol.