GBT1118, a voxelotor analog, protects red blood cells from damage during severe hypoxia.

A lack of objective metrics in Sickle Cell Disease (SCD) makes it difficult to assess individual patient therapy options or assess the effects of therapy. This is further complicated by mechanisms of action involving multiple interconnected effects, that combine to relieve SCD symptoms. In 2019, based on the increase in hemoglobin concentration observed in the HOPE trial, the Food and Drug Administration approved voxelotor (Oxbryta®, Global Blood Therapeutics) for SCD patients 12 years and older. The main mechanism of action for voxelotor was increased hemoglobin-oxygen affinity, but other mechanisms may apply. In this study, we assessed the effect of GBT1118, an Oxbryta analog, on hypoxia-induced lethal and sub-hemolytic red blood cell (RBC) membrane damage using RBC Mechanical Fragility (MF), a metric of existing membrane damage and prospective hemolysis. RBC MF was measured non-invasively using a proprietary system comprising an electromagnetic bead mill and fiberoptic spectrophotometry detection. Three cycles of severe hypoxia (<5% oxygenated hemoglobin) with follow-up reoxygenation resulted in a significant increase in RBC MF for all SCD (Hb-S >60%) samples. Supplementation with GBT1118 caused no significant changes in pre-hypoxia RBC MF. However, following GBT1118 treatment, cell stability showed significantly less degradation, as evidenced by a significantly smaller RBC MF increase after three cycles of hypoxia-reoxygenation. These findings indicate that GBT1118 prevents hypoxia-induced membrane damage in sickled RBC, in part by alternative mechanisms not associated with induced changes in hemoglobin-oxygen affinity.

Longitudinal assessment of adhesion to vascular cell adhesion molecule-1 at steady state and during vaso-occlusive crises in sickle cell disease.

Sickle cell disease (SCD) is characterized by frequent and unpredictable vaso-occlusive crises (VOCs). Sickle erythrocytes (SSRBCs) contribute to VOCs by participating in a series of adhesive events with blood cells and the vascular endothelium. Adhesion assays have been used to evaluate the relationship between SSRBC adhesion and SCD severity. We developed a standardized, clinical flow adhesion assay of whole blood to vascular cell adhesion molecule (FA-WB-VCAM). The objective of this study was to assess the variability and clinical predictive value of FA-WB-VCAM in a six-month longitudinal, observational study (ELIPSIS) in SCD subjects during at-home, steady-state and self-reported VOCs, and following VOC resolution. We observed a strong relationship between FA-WB-VCAM and SCD severity. Adhesion indices were significantly lower in SCD subjects on hydroxycarbamide and increased during VOCs; at-home VOCs had significantly higher FA-WB-VCAM than steady-state and contact VOCs. SCD subjects with a high frequency of self-reported VOCs had a pro-adhesive phenotype at steady state and were stratified into a high-adhesive phenotype cohort; two years prospectively we observed a higher frequency of VOCs in the high-adhesion cohort. This study supports stratifying SCD subjects based on steady-state FA-WB-VCAM and suggests that FA-WB-VCAM may be a plausible surrogate end-point for SCD severity.

Can red blood cell function assays assess response to red cell-modifying therapies?

BACKGROUND: Red blood cell (RBC)-modifying therapies have provided new opportunities for patients with sickle cell disease, although the absence of validated biomarkers of RBC function is a barrier to FDA approval and clinical adoption. Flow Adhesion (FA) and Mechanical Fragility (MF) biomarkers objectively stratify individuals with SCD into pro-adhesive vs pro-hemolytic phenotypes respectively, which may potentially help predict therapeutic responses. OBJECTIVE: A Phase 3 clinical trial to determine the effectiveness of vepoloxamer, an RBC-modifying therapy in sickle cell disease (SCD), failed to meet its primary clinical outcome. The aim of this study was to determine whether standardized flow adhesion and mechanical fragility bioassays could differentiate cellular level "responders" from "non-responders" to vepoloxamer treatment. METHODS: Standardized biomarkers of RBC function (adhesion and mechanical fragility) were utilized in this study to assess the effect of veploxamer on blood samples collected from SCD subjects and to determine whether our assays could differentiate cellular-level "responders" from "non-responders" to vepoloxamer treatment. A Wilcoxon signed-rank test was used to test for differences in adhesion in response to varying vepoloxamer treatments and a Wilcoxon Mann-Whitney test was used to assess differences in mechanical fragility, pre- and post-vepoloxamer treatment. A p-value<0.05 was considered significant. RESULTS: In this study, we report that in vitro treatment with vepoloxamer reduced adhesion by >75%in 54%of patient samples and induced changes in the membranes of sickle erythrocytes (SSRBCs) making sickle cells behave more like normal erythrocytes (AARBCs) in terms of their resistance to hemolysis. CONCLUSION: This study demonstrates that the standardized flow adhesion and mechanical fragility biomarkers described here may be useful tools to predict clinical responders to RBC-modifying therapies.

Flow adhesion of whole blood to P-selectin: a prognostic biomarker for vaso-occlusive crisis in sickle cell disease.

Blood cell adhesion to P-selectin and vascular cell adhesion molecule-1 (VCAM-1) contributes to the pathophysiology of vaso-occlusion crisis (VOC) events in individuals with sickle cell disease (SCD). We evaluated the use of standardized flow adhesion biomarkers in a six-month, 35-subjects longitudinal study (ELIPSIS). Flow adhesion of whole blood on P-selectin (FA-WB-Psel) and VCAM1 (FA-WB-VCAM), and of isolated white blood cells on P-selectin (FA-WBC-Psel) and VCAM-1 (FA-WBC-VCAM) were elevated on VOC days compared with non-VOC days, but only FA-WB-Psel reached statistical significance (P = 0·015). Optimal cut-off values were established with Cox regression models for FA-WB-Psel [46 cells/mm²; hazard ratio (HR): 2·3; 95% confidence interval (CI):1·4-4·0; P = 0·01] and FA-WB-VCAM (408 cells/mm², HR:1·8; 95% CI: 0·9-3·45; P = 0·01). A combined (FA-WB-Psel and FA-WB-VCAM) multimarker risk score was also significantly (P = 0·0006) correlated with VOC risk that was two-fold higher for intermediate and 5·64-fold higher for high score. The concordance (C)-index for the multimarker score was 0·63 in the six-month period (95% CI: 0·56-0·70), indicating a better ability to distinguish patient risk of VOC, compared to individual biomarkers FA-WB-VCAM (C-index: 0·57; 95% CI: 0·49-0·65) or FA-WB-Psel (C-index: 0·58; 95% CI: 0·53-0·62). The presented multimarker score can be used to risk-stratify individuals with SCD during their steady state into low, intermediate, and high-risk strata for self-reported VOCs. Such risk stratification could help focus healthcare resources more efficiently to maintiain health, personalize treatment selection to each patient's individual needs, and potentially reduce healthcare costs.

Individual variability in response to a single sickling event for normal, sickle cell, and sickle trait erythrocytes.

Hemoglobin S (Hb-S) polymerization is the primary event in sickle cell disease causing irreversible damage to red blood cell (RBC) membranes over repeated polymerization cycles. A single polymerization triggered by a hypoxic environment was reported to result in reversibly (upon reoxygenation) decreased RBC deformability and increased mechanical fragility (MF). Individualized responses have not been reported, although RBC fragility can vary significantly even among healthy individuals. This study evaluates individual variability in response to a single hypoxia-induced sickling event, through changes in RBC MF. Blood was drawn from 10 normal (AA), 11 sickle cell (SS), and 7 sickle trait (AS) subjects-with Hb-S fraction, osmotic fragility, and medical history also collected. Mechanical stress was applied using a bead mill at 50-Hz oscillation for 0.5-30 minutes. MF profiles here give percent hemolysis upon successive durations of stressing. MF was measured for AA, SS, and AS cells-each equilibrated (1) with air, (2) with nitrogen in an anaerobic chamber, and (3) with air after the hypoxic event. While AA subjects exhibited significantly different changes in fragility upon hypoxia, in all cases there was recovery to close to the initial MF values on reoxygenation. For AS subjects, recovery at reoxygenation was observed only in about half of the cases. Fragility of SS cells increased in hypoxia and decreased with reoxygenation, with significantly variable magnitude of recovery. The variability of response for individual AS and SS subjects indicates that some are potentially at higher risk of irreversible hypoxia-induced membrane damage.

Differences in bead-milling-induced hemolysis of red blood cells due to shape and size of oscillating bead.

BACKGROUND: Red blood cell (RBC) susceptibility to hemolysis - or fragility - can be profiled by subjecting a sample to progressive durations of mechanical stress and measuring hemolysis upon each. The ability to control stress application with multiple variable parameters can be useful in various areas of research. Bead milling, by oscillating an object in a blood sample, can offer control of parameters including oscillation force and frequency. OBJECTIVE: This work addresses the role of bead shape and size, for a given container, in potentially creating qualitatively as well as quantitatively different fluidic stresses in the sample. METHODS: Identical, diluted RBC samples were stressed via bead milling using different beads, with other parameters the same. Resulting hemolysis was plotted for several time increments in each case. RESULTS: For a cylindrical bead oscillating at a given frequency and force, bead length was a determinant of albumin's protective effect on RBC, as reflected by mechanical fragility. Compared to a sphere of same diameter, the protective effect was absent with shorter cylinders, whereas for longer ones it appeared enhanced. CONCLUSIONS: Bead milling based RBC fragility testing could present a useful tool for creating, and studying effects of different shear stress types in inducing hemolysis.

An approach to measuring RBC haemolysis and profiling RBC mechanical fragility.

Red blood cells (RBC) can be damaged by medical products, from storage or from disease. Haemolysis (cell rupture and haemoglobin release) is often a key indicator, with mechanical fragility (MF) offering the potential to assess sub-haemolytic damage as well. This article reports on a unique approach to measuring haemolysis, without the need for centrifugation or other sample separation. It also reports on employing that in measuring blood fragility (susceptibility to haemolysis) under shear stress, utilising an electromagnet to cause a bead to oscillate within a cartridge that contains the sample. Cycling between stressing and optical measurement of induced haemolysis at progressively increasing durations of stress provides a fragility profile. Sub-system-level testing shows high accuracy for the haemolysis measurements and fair consistency for MF profiling. Improving accuracy and precision of profiling is a current focus and a fully integrated and automated version of this system is under development.

RBC mechanical fragility as a direct blood quality metric to supplement storage time.

INTRODUCTION: Lengthy storage times and associated storage lesion can result in reduced red blood cell (RBC) efficacy, particularly dangerous for massively transfused patients. Today's inventory management makes storage times the de-facto metric of blood quality. However, RBC units' quality may vary because of time-independent factors. Mechanical fragility (MF) of RBC, reflecting sub-lethal cell damage, can potentially provide a more physiologically relevant predictor of cell's performance "in vivo." METHODS: Mechanical stress was applied using a bead mill (50 Hz) over durations varying from 0.5 to 60 minutes, or using ultrasound (40 W) with durations from 0.1 to 120 seconds. MF profiles were described in terms of percentage hemolysis following stresses of specified durations. RESULTS: RBC MF declined significantly in the presence of albumin, with albumin protecting membrane against damage from elevated temperature or from methyl-ß-cyclodextrin or diamide. MF profiles allowed detection of sub-lethal membrane damage caused by elevated temperature, to a greater extent than was reflected by autohemolysis. Different types of profiles for RBC damage were associated with MF changes at different stress intensities and potentially stress types. CONCLUSIONS: These findings indicate that MF profiles can provide a powerful and versatile tool for investigation of RBC, as well as a potential metric of RBC quality.

Structural and functional characterization of a cytochrome P450 2B4 F429H mutant with an axial thiolate-histidine hydrogen bond.

The structural basis of the regulation of microsomal cytochrome P450 (P450) activity was investigated by mutating the highly conserved heme binding motif residue, Phe429, on the proximal side of cytochrome P450 2B4 to a histidine. Spectroscopic, pre-steady-state and steady-state kinetic, thermodynamic, theoretical, and structural studies of the mutant demonstrate that formation of an H-bond between His429 and the unbonded electron pair of the Cys436 axial thiolate significantly alters the properties of the enzyme. The mutant lost >90% of its activity; its redox potential was increased by 87 mV, and the half-life of the oxyferrous mutant was increased ∼37-fold. Single-crystal electronic absorption and resonance Raman spectroscopy demonstrated that the mutant was reduced by a small dose of X-ray photons. The structure revealed that the δN atom of His429 forms an H-bond with the axial Cys436 thiolate whereas the εN atom forms an H-bond with the solvent and the side chain of Gln357. The amide of Gly438 forms the only other H-bond to the tetrahedral thiolate. Theoretical quantification of the histidine-thiolate interaction demonstrates a significant electron withdrawing effect on the heme iron. Comparisons of structures of class I-IV P450s demonstrate that either a phenylalanine or tryptophan is often found at the location corresponding to Phe429. Depending on the structure of the distal pocket heme, the residue at this location may or may not regulate the thermodynamic properties of the P450. Regardless, this residue appears to protect the thiolate from solvent, oxidation, protonations, and other deleterious reactions.

Similar donors-similar blood?

BACKGROUND: Red blood cell (RBC) storage lesions have been suggested as contributing factors to suboptimal clinical outcomes. While undesirable effects of storage are well documented, their clinical relevance is still debated. Focus on storage time as the sole determinant of RBC quality ignores the variability in cell properties that may depend on factors other than age. Mechanical fragility (MF) aggregately reflects many storage-related functional and structural changes. This study evaluates interdonor versus intradonor variability, throughout storage, of both MF and autohemolysis (AH). STUDY DESIGN AND METHODS: Thirteen uniformly manufactured RBC units were collected initially as whole blood from nonsmoking, group A+, male Caucasian research donors. Mechanical stress was applied using a bead mill with oscillation at 50 Hz over durations varying from 0.5 to 60 minutes. MF profiles were described in terms of percent hemolysis after stresses of specified durations. Two months later, 11 of the 13 donors returned and assays were performed using the same protocol to allow comparison of intradonor versus interdonor variation. RESULTS: At 5 days postcollection, RBC MF profiles exhibited marked interdonor variability (up to twofold) overall. Both autolysis and MF across all units increased during storage-with rates of these increases varying by up to 10-fold for certain MF variables. Especially high AH and MF were observed for an outlier donor (with p < 0.05), for whom follow-up revealed previously undisclosed hereditary hypertriglyceridemia (levels exceeding approx. 1000 mg/dL). CONCLUSIONS: RBCs, even from similar donors, vary significantly in levels and changes of both AH and MF, the clinical significance of which must still be ascertained. While further study is needed, donors with severe hypertriglyceridemia may not be appropriate as blood donors due to the unacceptable level of hemolysis observed during storage of our affected study subject.

Stabilization and spectroscopic characterization of the dioxygen complex of wild-type cytochrome P4502B4 (CYP2B4) and its distal side E301Q, T302A and proximal side F429H mutants at subzero temperatures.

Mammalian cytochrome P450 2B4 (CYP2B4) is a phenobarbital-inducible rabbit hepatic monooxygenase that catalyzes the N-demethylation of benzphetamine and metabolism of numerous other compounds. To probe the interactions of the heme environment and bound benzphetamine with the dioxygen (O2) complex of CYP2B4, homogeneous O2 complexes of the wild-type enzyme and three mutants at sites of conserved amino acids, two on the heme distal side (T302A and E301Q) and one on the proximal side (F429H), have been prepared and stabilized at ~-50°C in mixed solvents (60-70% v/v glycerol). We report that the magnetic circular dichroism and electronic absorption spectra of wild-type oxyferrous CYP2B4, in the presence and absence of substrate, are quite similar to those of the dioxygen complex of bacterial cytochrome P450-CAM (CYP101). However, the oxyferrous complexes of the T302A and E301Q CYP2B4 mutants have significantly perturbed electronic structure (~4 nm and ~3 nm red-shifted Soret features, respectively) compared to that of the wild-type oxyferrous complex. On the other hand, the heme proximal side mutant, CYP2B4 F429H, undergoes relatively facile conversion to a partially (~50%) denatured (P420) form upon reduction. The structural changes in the heme pocket environments of the CYP2B4 mutants that lead to the spectroscopic distinctions reported herein can be related to the differences in oxidation activities of wild-type CYP2B4 and its E301Q, T302A and F429H mutants.

Distal end of 105-125 loop--a putative reductase binding domain of phthalate dioxygenase.

The phthalate dioxygenase system consists of the dioxygenase, PDO, which contains a Rieske [2Fe-2S] center and a Fe(II)-mononuclear center, and the reductase, PDR. Involvement of the distal end of the 105-125 loop of PDO in its interaction with PDR was tested by substituting charged residues in the loop with alanines and by replacing the conserved tryptophan-94. Compared to wild-type PDO, all variants had lower catalytic activity and the Rieske centers were reduced more slowly by reduced PDR. The rates of oxidation of the Rieske centers by oxygen, which represent electron transfer between the Rieske and mononuclear centers, were essentially unaffected. These results suggest that positively charged residues of the distal end of the 105-125 loop are collectively involved in PDR binding with the PDO. Contrary to expectations, Trp94 variants were not directly involved in electron transfer between PDR and PDO. The tryptophan appears to have mainly a structural role, apparently preserving the hydrophilic environment of the Rieske center.

Similar enzymes, different structures: phthalate dioxygenase is an alpha3alpha3 stacked hexamer, not an alpha3beta3 trimer like "normal" Rieske oxygenases.

Phthalate dioxygenase (PDO) is a member of a class of bacterial oxygenases that contain both Rieske [2Fe-2S] and Fe(II) mononuclear centers. Recent crystal structures of several Rieske dioxygenases showed that they exist as alpha(3)beta(3) multimers with subunits arranged head-to-tail in alpha and beta stacked planar rings. The structure of PDO, which consists of only alpha-subunits, remains to be solved. Although similar to other Rieske dioxygenases in many aspects, PDO was shown to differ in the mechanism of catalysis. Gel filtration and analytical centrifugation experiments, supplemented with mass spectrometric analysis (both ESI-MS and ESI-GEMMA), in this work showed a hexameric arrangement of subunits in the PDO multimer. Our proposed model for the subunit arrangement in PDO postulates two alpha(3) planar rings one on top the other, similar to the alpha(3)beta(3) arrangement in other Rieske dioxygenases. Unlike other Rieske dioxygenases, this arrangement brings two Rieske and two mononuclear centers, all on separate subunits, into proximity, allowing their cooperation for catalysis. Potential reasons necessitating this unusual structural arrangement are discussed.

High levels of expression of the iron-sulfur proteins phthalate dioxygenase and phthalate dioxygenase reductase in Escherichia coli.

Phthalate dioxygenase (PDO), a hexamer with one Rieske-type [2Fe-2S] and one Fe (II)-mononuclear center per monomer, and its reductase (PDR), which contains flavin mononucleotide and a plant-type ferredoxin [2Fe-2S] center, are expressed by Burkholderia cepacia at approximately 30mg of crude PDO and approximately 1mg of crude PDR per liter of cell culture when grown with phthalate as the main carbon source. A high level expression system in Escherichia coli was developed for PDO and PDR. Optimization relative to E. coli cell line, growth parameters, time of induction, media composition, and iron-sulfur additives resulted in yields of about 1g/L for PDO and about 0.2g/L for PDR. Protein expression was correlated to the increase in pH of the cell culture and exhibited a pronounced (variable from 5 to 20h) lag after the induction. The specific activity of purified PDO did not depend on the pH of the cell culture when harvested. However, when the pH of the culture reached 8.5-9, a large fraction of the PDR that was expressed lacked its ferredoxin domain, presumably because of proteolysis. Termination of growth while the pH of the cell culture was <8 decreased the fraction of proteolyzed enzyme, whereas yields of the unclipped PDR were only marginally lower. Overall, changes in pH of the cell culture were found to be an excellent indicator of the overall level of native protein expression. Its monitoring allowed the real time tracking of the protein expression and made it possible to tailor the expression times to achieve a combination of high quality and high yield of protein.

The "bridging" aspartate 178 in phthalate dioxygenase facilitates interactions between the Rieske center and the iron(II)--mononuclear center.

Phthalate dioxygenase (PDO) and its reductase are parts of a two-component Rieske dioxygenase system that initiates the aerobic breakdown of phthalate by forming cis-4,5-dihydro-4,5-dihydroxyphthalate (DHD). Aspartate D178 in PDO, located near its ferrous mononuclear center, is highly conserved among Rieske dioxygenases. The analogous aspartate has been implicated in electron transfer between the mononuclear iron and Rieske center in naphthalene dioxygenase [Parales et al. (1999) J. Bacteriol. 181, 1831-1837] and in substrate binding and oxygen reactivity in anthranilate dioxygenase [Beharry et al. (2003) Biochemistry 42, 13625-13636]. The effects of substituting D178 in PDO with alanine or asparagine on the reactivity of the Rieske centers, phthalate hydroxylation, and coupling of Rieske center oxidation to DHD formation were studied previously [Pinto et al. (2006) Biochemistry 45, 9032-9041]. This work describes effects that D178N and D178A substitutions have on the interactions between the Rieske and mononuclear centers in PDO. The mutations affected protonation of the Rieske center histidine and conformation of subunits within the PDO multimer to create a more open structure with more solvent-accessible Rieske centers. When the Rieske centers in PDO were oxidized, D178N and D178A substitutions disrupted communication between the Rieske and Fe-mononuclear centers. This was shown by the lack of perturbations of the UV-vis spectra on phthalate binding to the D178N and D178A variants, as opposed to that observed in WT PDO. However, when the Rieske center was in the reduced state, communication between the centers was not disrupted. Phthalate binding similarly affected the rates of oxidation of the reduced Rieske center in both WT and mutant PDO. Nitric oxide binding at the Fe(II)-mononuclear center, as detected by EPR spectrometry of the Fe(II) nitrosyl complex, was regulated by the redox state of the Rieske center. When the Rieske center was oxidized in either WT or D178N PDO, NO bound to the mononuclear iron in the presence or absence of phthalate. However, when the Rieske center was reduced, NO bound only when phthalate was present. These findings are discussed in terms of the "communication functions" performed by the bridging Asp-178.

Substitutions of the "bridging" aspartate 178 result in profound changes in the reactivity of the Rieske center of phthalate dioxygenase.

Phthalate dioxygenase (PDO) and its reductase (PDR) are parts of a two-component Rieske oxygenase system that initiates the aerobic breakdown of phthalate by forming cis-4,5-dihydro-4,5-dihydroxyphthalate. Aspartate D178 in PDO, which lies between the Rieske [2Fe-2S] center of one subunit and the mononuclear center of the adjacent subunit, is highly conserved among the Rieske dioxygenases. The analogous aspartate has been implicated in electron transfer in naphthalene dioxygenase and in substrate binding and oxygen reactivity in anthranilate dioxygenase. Substitution of D178 with alanine or asparagine in PDO resulted in proteins with significantly increased Fe(II) dissociation constants. The rates of oxidation of the reduced Rieske centers in D178A and D178N were decreased by more than 10(4)-fold; only part of the loss of activity can be attributed to depletion of iron from the mononuclear centers. Reduction of PDO by reduced PDR was also slower in the D178A and D178N variants. Observed decreases in turnover rates of D178A and D178N compared to that of wild-type (WT) PDO (>10(2)-fold) can be ascribed to the cumulative effect of the low intrinsic iron content of the D178A and D178N mutants and the combination of the decreased rates of Rieske center reduction and oxidation. The coupling of dihydrodiol formation approached 100% in WT PDO but was only approximately 16% in D178A and approximately 7% in D178N. In single-turnover experiments, very small amounts of DHD were produced by D178A and D178N "as purified". The presence of saturating amounts of ferrous ion improved coupling to nearly 100% for the D178N variant but only slightly improved coupling for D178A. Thus, although hydroxylation is still possible in the variants, the reactions are largely uncoupled due to slow intramolecular electron transfer rates and the apparent weak binding of iron at the mononuclear centers.

Chemistry of the catalytic conversion of phthalate into its cis-dihydrodiol during the reaction of oxygen with the reduced form of phthalate dioxygenase.

The phthalate dioxygenase system, a Rieske non-heme iron dioxygenase, catalyzes the dihydroxylation of phthalate to form the 4,5-dihydro-cis-dihydrodiol of phthalate (DHD). It has two components: phthalate dioxygenase (PDO), a multimer with one Rieske-type [2Fe-2S] and one mononuclear Fe(II) center per monomer, and a reductase (PDR) that contains flavin mononucleotide (FMN) and a plant-type ferredoxin [2Fe-2S] center. This work shows that product formation in steady-state reactions is tightly coupled to electron delivery, with 1 dihydrodiol (DHD) of phthalate formed for every 2 electrons delivered from NADH. However, in reactions of reduced PDO with O(2), only about 0.5 DHD is formed per Rieske center that becomes oxidized. Although the product forms rapidly, its release from PDO is slow in these reactions with oxygen that do not include reductase and NADH. EPR data show that, at the completion of the oxidation, iron in the mononuclear center remains in the ferrous state. In contrast, naphthalene dioxygenase (NDO) [Wolfe, M. D., Parales, J. V., Gibson, D. T., and Lipscomb, J. D. (2001) J. Biol. Chem. 276, 1945-1953] and benzoate dioxygenase (BZDO) [Wolfe, M. D., Altier, D. J., Stubna, A., Popescu, C. V., Munck, E., and Lipscomb, J. D. (2002) Biochemistry, 41, 9611-9626], related Rieske non-heme iron dioxygenases, form 1 DHD per Rieske center oxidized, and the mononuclear center iron ends up ferric. Thus, both electrons from reduced NDO and BZDO monomers are used to form the product, whereas only the reduced Rieske centers in PDO become oxidized during production of DHD. This emphasizes the importance of PDO subunit interaction in catalysis. Electron redistribution was practically unaffected by the presence of oxidized PDR. A scheme is presented that emphasizes some of the differences in the mechanisms involved in substrate hydroxylation employed by PDO and either NDO or BZDO.

Rates of the phthalate dioxygenase reaction with oxygen are dramatically increased by interactions with phthalate and phthalate oxygenase reductase.

The phthalate dioxygenase system, which catalyzes the dihydroxylation of phthalate to form its cis-dihydrodiol (DHD), has two components: phthalate dioxygenase (PDO), a multimer with one Rieske-type [2Fe-2S] and one Fe(II) center per monomer, and phthalate dioxygenase reductase (PDR), which contains flavin mononucleotide (FMN) and a plant-like ferredoxin [2Fe-2S] center. PDR is responsible for transferring electrons from NADH to the Rieske center of PDO, and the Rieske center supplies electrons to the mononuclear center for the oxygenation of substrate. Reduced PDO (PDO(red)) that lacks Fe(II) at the mononuclear metal site (PDO-APO) reacts slowly with O(2) (1.4 x 10(-3) s(-1) at 125 microM O(2) and 22 degrees C), presumably in a direct reaction with the Rieske center. Binding of phthalate and/or PDR(ox) to reduced PDO-APO increases the reactivity of the Rieske center with O(2). When no PDR or phthalate is present, the oxidation of the Rieske center in native PDO(red) [which contains Fe(II) at the mononuclear site] occurs in two phases (approximately 1 and 0.1 s(-1) at 125 mM O(2), 23 degrees C), both much faster than in the absence of Fe(II), presumably because in this case O(2) reacts at the mononuclear Fe(II). Addition of PDR(ox) to native PDO(red) resulted in a large fraction of the Rieske center being oxidized at 5 s(-1), and the addition of phthalate resulted in about 70% of the reaction proceeding at 42 s(-1). With both PDR(ox) and phthalate present, most of the PDO(red) (approximately 80-85%) oxidizes at 42 s(-1), with the remaining oxidizing at approximately 5 s(-1). Thus, the binding of phthalate or PDR(ox) to PDO(red) each results in greater reactivity of PDO with O(2). The presence of both the substrate and PDR was synergistic, making PDO fully catalytically active. A model that explains the observed effects is presented and discussed in terms of PDO subunit cooperativity. It is proposed that, during oxidation of reduced PDO, each of two Rieske centers on separate subunits transfers an electron to the Fe(II) mononuclear center on a third subunit. This explanation is consistent with the observed multiphasic kinetics of the oxidation of the Rieske center and is being further tested by product analysis experiments.

Detergent modulation of electron and proton transfer reactions in bovine cytochrome c oxidase.

The effect of detergents on electron and proton transfer in bovine cytochrome c oxidase was studied using steady-state and transient-state methods. Cytochrome c oxidase in lauryl maltoside has high maximal turnover (TN(max)=400 s(-1)), whereas activity is low (TN(max)=10 s(-1)) in Triton X-100. Single turnover studies of intramolecular electron transfer show similar rates in either detergent. Transient proton uptake experiments show the oxidase in lauryl maltoside consumes 1.8+/-0.3 H(+)/aa(3) during either partial reduction of the oxidase or reaction of fully reduced enzyme with O(2). However, the oxidase in Triton X-100 consumes 2.6+/-0.4 H(+)/aa(3) during partial reduction and 1.0+/-0.2 H(+)/aa(3) in the O(2) reaction. Absorption spectra recorded during turnover show that the enzyme undergoes activation in lauryl maltoside, but does not activate in Triton X-100. We propose that cytochrome c oxidase in different detergents allows access to different sites of protonation, which in turn influences steady-state activity.