Probing the Protein-Excipient Interaction in the Orally Delivered Protein by Solid-State Hydrogen-Deuterium Exchange Mass Spectrometry and Molecular Dynamics.

The oral administration of protein therapeutics in solid dosage form is gaining popularity due to its benefits, such as improved medication adherence, convenience, and ease of use for patients compared to traditional parental delivery. However, formulating oral biologics presents challenges related to pH barriers, enzymatic breakdown, and poor bioavailability. Therefore, understanding the interaction between excipients and protein therapeutics in the solid state is crucial for formulation development. In this Letter, we present a case study focused on investigating the role of excipients in protein aggregation during the production of a solid dosage form of a single variable domain on a heavy chain (VHH) protein. We employed solid-state hydrogen-deuterium exchange coupled with mass spectrometry (ssHDX-MS) at both intact protein and peptide levels to assess differences in protein-excipient interactions between two formulations. ssHDX-MS analysis revealed that one formulation effectively prevents protein aggregation during compaction by blocking ß-sheets across the VHH protein, thereby preventing ß-sheet-ß-sheet interactions. Spatial aggregation propensity (SAP) mapping and cosolvent simulation from molecular dynamics (MD) simulation further validated the protein-excipient interaction sites identified through ssHDX-MS. Additionally, the MD simulation demonstrated that the interaction between the VHH protein and excipients involves hydrophilic interactions and/or hydrogen bonding. This novel approach holds significant potential for understanding protein-excipient interactions in the solid state and can guide the formulation and process development of orally delivered protein dosage forms, ultimately enhancing their efficacy and stability.

A Mechanistic Understanding of Monoclonal Antibody Interfacial Protection by Hydrolytically Degraded Polysorbate 20 and 80 under IV Bag Conditions.

PURPOSE: Polysorbates (PS) contain polyoxyethylene (POE) sorbitan/isosorbide fatty acid esters that can partially hydrolyze over time in liquid drug products to generate degradants and a remaining intact PS fraction with a modified ester distribution. The degradants are composed of free fatty acids (FFAs) --primarily lauric acid for PS20 and oleic acid for PS80-- and POE head groups. We previously demonstrated that under IV bag agitation conditions, mAb1 (a surface-active IgG4) aggregation increased with increasing amounts of degradants for PS20 but not for PS80. The purpose of this work is to understand the mechanism behind this observation. METHODS: The surface tension of the remaining intact PS fraction without degradants was modeled and compared with that of enzymatically degraded PS solutions. Next, mAb1 aggregation in saline was measured in the presence of laurate and oleate salts during static storage. Lastly, colloidal and conformational stability of mAb1 in the presence of these salts was investigated through differential scanning fluorimetry and dynamic light scattering under IV bag solution conditions. RESULTS: The surface tension was primarily influenced by FFAs rather than the modified ester distribution of the remaining intact PS. MAb1 bulk aggregation increased in the presence of laurate but not oleate salts. Both salt types increased the melting temperature of mAb1 indicating FFA-mAb1 interactions. However, only laurate salt increased mAb1 self-association potentially explaining the higher aggregation propensity in its presence. CONCLUSION: Our results help explain the observed differences between hydrolytically degraded PS20 and PS80 in affecting mAb1 aggregation under IV bag agitation conditions.

An Interlaboratory Study to Identify Potential Visible Protein-Like Particle Standards.

Visible protein-like particle standards may improve visual inspection and/or appearance testing practices used in the biotechnology industry. They may improve assay performance resulting in better alignment and more standardized training among different companies. The National Institute of Standards and Technology (NIST) has conducted an interlaboratory study to test whether the standards under development mimic typical proteinaceous particles found in biotherapeutics and if they can be implemented during the visual inspection process. Fourteen organizations from industry and government have participated. A total of 20 labs from these 14 organizations participated with analysts from 6 formulation, 7 analytical, 4 quality control, and 3 manufacturing labs. The circulated samples consisted of abraded ethylene tetrafluoroethylene (ETFE) particles or photolithographic particles. The results consist of qualitative ratings, which varied substantially among organizations and within labs. Polydisperse ETFE particle suspensions, containing particles enriched in greater than 150 µm in size, were rated more favorably than the photolithographic particles by formulation and analytical scientists. The largest monodisperse photolithographic particles (approximately 300 µm in size) were favored equally compared to ETFE by all scientists. Solution modifications to decrease the settling rate or to alter optical properties of the ETFE solutions yielded lower ratings by the analysts. Both particle types received mixed ratings for their usability and for their application for visual inspection and for training purposes. Industry feedback will assist NIST in developing reference material(s) for visible protein-like particles.

Evaluating a Modified High Purity Polysorbate 20 Designed to Reduce the Risk of Free Fatty Acid Particle Formation.

PURPOSE: To evaluate a modified high purity polysorbate 20 (RO HP PS20)-with lower levels of stearate, palmitate and myristate esters than the non-modified HP PS20-as a surfactant in biopharmaceutical drug products (DP). RO HP PS20 was designed to provide functional equivalence as a surfactant while delaying the onset of free fatty acid (FFA) particle formation upon hydrolytic degradation relative to HP PS20. METHODS: Analytical characterization of RO HP PS20 raw material included fatty acid ester (FAE) distribution, higher order ester (HOE) fraction, FFA levels and trace metals. Functional assessments included 1) vial and intravenous bag agitation; 2) oxidation via a placebo and methionine surrogate study; and 3) hydrolytic PS20 degradation studies to evaluate FFA particle formation with and without metal nucleation. RESULTS: Interfacial protection and oxidation propensity were comparable between the two polysorbates. Upon hydrolytic degradation, FFA particle onset was delayed in RO HP PS20. The delay was more pronounced when HOEs of PS20 were preferentially degraded. Furthermore, the hydrolytic degradants of RO HP PS20 formed fewer particles in the presence of spiked aluminum. CONCLUSION: This work highlights the criticality of having tighter control on long chain FAE levels of PS20 to reduce the occurrence of FFA particle formation upon hydrolytic degradation and lower the variability in its onset. By simultaneously meeting compendial PS20 specifications while narrowing the allowable range for each FAE and shifting its composition towards the shorter carbon chain species, RO HP PS20 provides a promising alternative to HP PS20 for biopharmaceutical DPs.

A Random Forest Approach for Counting Silicone Oil Droplets and Protein Particles in Antibody Formulations Using Flow Microscopy.

PURPOSE: To evaluate a random forest model that counts silicone oil droplets and non-silicone oil particles in protein formulations with large class imbalance. METHODS: In this work, we present a novel approach for automated image analysis of flow microscopy data based on random forest classification enabling rapid analysis of large data sets. The random forest approach overcomes many of the limitations of traditional classification schemes derived from simple filters or regression models. In particular, the approach does not require a priori selection of important morphology parameters. RESULTS: We analyzed silicone oil droplets and non-silicone oil particles observed in four model systems with protein concentrations of 20, 50 and 125 mg/mL. Filters based on random forests achieve higher classification accuracies when compared to regression based filters. Additionally, we showcase a procedure that allows for accurate counting of particles ≥1 µm. CONCLUSIONS: Our method is generally applicable for classification and counting of different classes of particles as long as class morphologies are differentially expressed.

Identification of Subvisible Particles in Biopharmaceutical Formulations Using Raman Spectroscopy Provides Insight into Polysorbate 20 Degradation Pathway.

PURPOSE: To study composition and heterogeneity of insoluble subvisible particles in Mab formulations resulting from degradation of polysorbate 20 and to develop a better understanding of the mechanisms of polysorbate degradation leading to particle formation. METHODS: In this study, we exploit the potential of Raman microscopy for chemical identification of particles in monoclonal antibody formulations. Through a combination of experiments and density functional theory (DFT) calculations, we identified unique spectral marker bands for insoluble degradation products of polysorbate 20. We first applied our methodology to identify particles in model systems containing complex mixtures of fatty acids and then to subvisible particles in antibody formulations stored at 5°C for several years. RESULTS: Most of the subvisible particles identified were comprised of mixtures of fatty acids with no observable signal from fatty acid esters consistent with hydrolysis being the predominant degradation mechanism leading to particulate formation under these storage conditions. CONCLUSIONS: Our methodology is generally applicable for identification of particles in antibody formulations and, in particular, has the potential to give detailed information about particle heterogeneity and insight into mechanistic aspects of particle formation.

Stability Comparison Between Microglassification and Lyophilization Using a Monoclonal Antibody.

Producing solid-state formulations of biologics remains a daunting task despite the prevalent use of lyophilization and spray drying technologies in the biopharmaceutical industry. The challenges include protein stability (temperature stresses), high capital costs, particle design/controllability, shortened processing times and manufacturing considerations (scalability, yield improvements, aseptic operation, etc.). Thus, scientists/engineers are constantly working to improve existing methodologies and exploring novel dehydration/powder-forming technologies. Microglassification™ is a dehydration technology that uses solvent extraction to rapidly dehydrate protein formulations at ambient temperatures, eliminating the temperature stress experienced by biologics in traditional lyophilization and spray drying methods. The process results in microparticles that are spherical, dense, and chemically stable. In this study, we compared the molecular stability of a monoclonal antibody formulation processed by lyophilization to the same formulation processed using Microglassification™. Both powders were placed on stability for 3 months at 40 °C and 6 months at 25 °C. Both dehydration methods showed similar chemical stability, including percent monomer, charge variants, and antigen binding. These results show that Microglassification™ is viable for the production of stable solid-state monoclonal antibody formulations.

Monitoring of Visible Particles in Parenteral Products by Manual Visual Inspection-Reassessing Size Threshold and Other Particle Characteristics that Define Particle Visibility.

Visible particles are a critical quality attribute for parenteral products and must be monitored. A carefully designed, executed, and controlled drug product manufacturing process including a final 100 % visual inspection and appropriate end-product controls ensures that visible particles are consistently minimized and demonstrates that the injectable DP is practically free from visible particles. Visual inspection, albeit appearing as a simple analytical procedure, requires several technical and operational controls to ensure adequate performance. To gather new data on particle visibility and shed light on this decade-old challenge, a multi-company blinded visual inspection threshold study was conducted. A major goal of the study was visual assessment of several particle types of different sizes in small volume vials, as a challenging configuration for visual inspection, across 9 biopharmaceutical companies in order to determine the visibility limit. The study results provide key insights into limitations and challenges of visual inspection, namely, no universal visibility limit can be applied to all particle types as the detectability varies with particle type, number, and size. The study findings underscore the necessity of setting realistic expectations on size-based visibility limits in visual inspection, robust procedures for analyst training and qualification, and harmonization of guidelines globally.

Evaluating Clinical Safety and Analytical Impact of Subvisible Silicone Oil Particles in Biopharmaceutical Products.

Silicone oil is a commonly used lubricant in pre-filled syringes (PFSs) and can migrate over time into solution in the form of silicone oil particles (SiOPs). The presence of these SiOPs can result in elevated subvisible particle counts in PFS drug products compared to other drug presentations such as vials or cartridges. Their presence in products presents analytical challenges as they complicate quantitation and characterization of other types of subvisible particles in solution. Previous studies have suggested that they can potentially act as adjuvant resulting in potential safety risks for patients. In this paper we present several analytical case studies describing the impact of the presence of SiOPs in biotherapeutics on the analysis of the drug as well as clinical case studies examining the effect of SiOPs on patient safety. The analytical case studies demonstrate that orthogonal techniques, especially flow imaging, can help differentiate SiOPs from other types of particulate matter. The clinical case studies showed no difference in the observed patient safety profile across multiple drugs, patient populations, and routes of administration, indicating that the presence of SiOPs does not impact patient safety.

A unique iron-sulfur cluster is crucial for oxygen tolerance of a [NiFe]-hydrogenase.

Hydrogenases are essential for H(2) cycling in microbial metabolism and serve as valuable blueprints for H(2)-based biotechnological applications. However, most hydrogenases are extremely oxygen sensitive and prone to inactivation by even traces of O(2). The O(2)-tolerant membrane-bound [NiFe]-hydrogenase of Ralstonia eutropha H16 is one of the few examples that can perform H(2) uptake in the presence of ambient O(2). Here we show that O(2) tolerance is crucially related to a modification of the internal electron-transfer chain. The iron-sulfur cluster proximal to the active site is surrounded by six instead of four conserved coordinating cysteines. Removal of the two additional cysteines alters the electronic structure of the proximal iron-sulfur cluster and renders the catalytic activity sensitive to O(2) as shown by physiological, biochemical, spectroscopic and electrochemical studies. The data indicate that the mechanism of O(2) tolerance relies on the reductive removal of oxygenic species guided by the unique architecture of the electron relay rather than a restricted access of O(2) to the active site.

A Novel Technique to Assess Drug Substance Particle Size in a Complex Inhaled Formulation.

Dry powder inhalers, comprising an active pharmaceutical ingredient (API) and carrier excipients, are often used in the delivery of pulmonary drugs. The stability of the API particle size within a formulation blend is a critical attribute for aerodynamic performance but can be challenging to measure. The presence of excipients, typically at concentrations much higher than API, makes measurement by laser diffraction very difficult. This work introduces a novel laser diffraction approach that takes advantage of solubility differences between the API and excipients. The method allows insight into the understanding of drug loading effects on API particle stability of the drug product. Lower drug load formulations show better particle size stability compared with high drug load formulations, likely due to reduced cohesive interactions.

Experimental quantification of electrostatics in X-H···π hydrogen bonds.

Hydrogen bonds are ubiquitous in chemistry and biology. The physical forces that govern hydrogen-bonding interactions have been heavily debated, with much of the discussion focused on the relative contributions of electrostatic vs quantum mechanical effects. In principle, the vibrational Stark effect, the response of a vibrational mode to electric field, can provide an experimental method for parsing such interactions into their electrostatic and nonelectrostatic components. In a previous study we showed that, in the case of relatively weak O-H···π hydrogen bonds, the O-H bond displays a linear response to an electric field, and we exploited this response to demonstrate that the interactions are dominated by electrostatics (Saggu, M.; Levinson, N. M.; Boxer, S. G. J. Am. Chem. Soc.2011, 133, 17414-17419). Here we extend this work to other X-H···π interactions. We find that the response of the X-H vibrational probe to electric field appears to become increasingly nonlinear in the order O-H < N-H < S-H. The observed effects are consistent with differences in atomic polarizabilities of the X-H groups. Nonetheless, we find that the X-H stretching vibrations of the model compounds indole and thiophenol report quantitatively on the electric fields they experience when complexed with aromatic hydrogen-bond acceptors. These measurements can be used to estimate the electrostatic binding energies of the interactions, which are found to agree closely with the results of energy calculations. Taken together, these results highlight that with careful calibration vibrational probes can provide direct measurements of the electrostatic components of hydrogen bonds.

Multi-attribute Raman spectroscopy (MARS) for monitoring product quality attributes in formulated monoclonal antibody therapeutics.

Rapid release of biopharmaceutical products enables a more efficient drug manufacturing process. Multi-attribute methods that target several product quality attributes (PQAs) at one time are an essential pillar of the rapid-release strategy. The novel, high-throughput, and nondestructive multi-attribute Raman spectroscopy (MARS) method combines Raman spectroscopy, design of experiments, and multivariate data analysis (MVDA). MARS allows the measurement of multiple PQAs for formulated protein therapeutics without sample preparation from a single spectroscopic scan. Variable importance in projection analysis is used to associate the chemical and spectral basis of targeted PQAs, which assists in model interpretation and selection. This study shows the feasibility of MARS for the measurement of both protein purity-related and formulation-related PQAs; measurements of protein concentration, osmolality, and some formulation additives were achieved by a generic multiproduct model for various protein products containing the same formulation components. MARS demonstrates the potential to be a powerful methodology to improve the efficiency of biopharmaceutical development and manufacturing, as it features fast turnaround time, good robustness, less human intervention, and potential for automation.

An Interlaboratory Comparison on the Characterization of a Sub-micrometer Polydisperse Particle Dispersion.

The measurement of polydisperse protein aggregates and particles in biotherapeutics remains a challenge, especially for particles with diameters of ≈ 1 µm and below (sub-micrometer). This paper describes an interlaboratory comparison with the goal of assessing the measurement variability for the characterization of a sub-micrometer polydisperse particle dispersion composed of five sub-populations of poly(methyl methacrylate) (PMMA) and silica beads. The study included 20 participating laboratories from industry, academia, and government, and a variety of state-of-the-art particle-counting instruments. The received datasets were organized by instrument class to enable comparison of intralaboratory and interlaboratory performance. The main findings included high variability between datasets from different laboratories, with coefficients of variation from 13 % to 189 %. Intralaboratory variability was, on average, 37 % of the interlaboratory variability for an instrument class and particle sub-population. Drop-offs at either end of the size range and poor agreement on maximum counts of particle sub-populations were noted. The mean distributions from an instrument class, however, showed the size-coverage range for that class. The study shows that a polydisperse sample can be used to assess performance capabilities of an instrument set-up (including hardware, software, and user settings) and provides guidance for the development of polydisperse reference materials.

Biosynthesis of (bacterio)chlorophylls: ATP-dependent transient subunit interaction and electron transfer of dark operative protochlorophyllide oxidoreductase.

Dark operative protochlorophyllide oxidoreductase (DPOR) catalyzes the light-independent two-electron reduction of protochlorophyllide a to form chlorophyllide a, the last common precursor of chlorophyll a and bacteriochlorophyll a biosynthesis. During ATP-dependent DPOR catalysis the homodimeric ChlL(2) subunit carrying a [4Fe-4S] cluster transfers electrons to the corresponding heterotetrameric catalytic subunit (ChlN/ChlB)(2), which also possesses a redox active [4Fe-4S] cluster. To investigate the transient interaction of both subcomplexes and the resulting electron transfer reactions, the ternary DPOR enzyme holocomplex comprising subunits ChlN, ChlB, and ChlL from the cyanobacterium Prochlorococcus marinus was trapped as an octameric (ChlN/ChlB)(2)(ChlL(2))(2) complex after incubation with the nonhydrolyzable ATP analogs adenosine 5'-(gamma-thio)triphosphate, adenosine 5'-(beta,gamma-imido)triphosphate, or MgADP in combination with AlF(4)(-). Additionally, a mutant ChlL(2) protein, with a deleted Leu(153) in the switch II region also allowed for the formation of a stable octameric complex. Furthermore, efficient complex formation required the presence of protochlorophyllide. Electron paramagnetic resonance spectroscopy of ternary DPOR complexes revealed a reduced [4Fe-4S] cluster located on ChlL(2), indicating that complete ATP hydrolysis is a prerequisite for intersubunit electron transfer. Circular dichroism spectroscopic experiments indicated nucleotide-dependent conformational changes for ChlL(2) after ATP binding. A nucleotide-dependent switch mechanism triggering ternary complex formation and electron transfer was concluded. From these results a detailed redox cycle for DPOR catalysis was deduced.

Direct measurements of electric fields in weak OH···π hydrogen bonds.

Hydrogen bonds and aromatic interactions are of widespread importance in chemistry, biology, and materials science. Electrostatics play a fundamental role in these interactions, but the magnitude of the electric fields that support them has not been quantified experimentally. Phenol forms a weak hydrogen bond complex with the π-cloud of benzene, and we used this as a model system to study the role of electric fields in weak OH···π hydrogen bonds. The effects of complex formation on the vibrational frequency of the phenol OH or OD stretches were measured in a series of benzene-based aromatic solvents. Large shifts are observed and these can be converted into electric fields via the measured vibrational Stark effect. A comparison of the measured fields with quantum chemical calculations demonstrates that calculations performed in the gas phase are surprisingly effective at capturing the electrostatics observed in solution. The results provide quantitative measurements of the magnitude of electric fields and electrostatic binding energies in these interactions and suggest that electrostatics dominate them. The combination of vibrational Stark effect (VSE) measurements of electric fields and high-level quantum chemistry calculations is a general strategy for quantifying and characterizing the origins of intermolecular interactions.

Characterization and crystallization of mouse aldehyde oxidase 3: from mouse liver to Escherichia coli heterologous protein expression.

Aldehyde oxidase (AOX) is characterized by a broad substrate specificity, oxidizing aromatic azaheterocycles, such as N¹-methylnicotinamide and N-methylphthalazinium, or aldehydes, such as benzaldehyde, retinal, and vanillin. In the past decade, AOX has been recognized increasingly to play an important role in the metabolism of drugs through its complex cofactor content, tissue distribution, and substrate recognition. In humans, only one AOX gene (AOX1) is present, but in mouse and other mammals different AOX homologs were identified. The multiple AOX isoforms are expressed tissue-specifically in different organisms, and it is believed that they recognize distinct substrates and carry out different physiological tasks. AOX is a dimer with a molecular mass of approximately 300 kDa, and each subunit of the homodimeric enzyme contains four different cofactors: the molybdenum cofactor, two distinct [2Fe-2S] clusters, and one FAD. We purified the AOX homolog from mouse liver (mAOX3) and established a system for the heterologous expression of mAOX3 in Escherichia coli. The purified enzymes were compared. Both proteins show the same characteristics and catalytic properties, with the difference that the recombinant protein was expressed and purified in a 30% active form, whereas the native protein is 100% active. Spectroscopic characterization showed that FeSII is not assembled completely in mAOX3. In addition, both proteins were crystallized. The best crystals were from native mAOX3 and diffracted beyond 2.9 Å. The crystals belong to space group P1, and two dimers are present in the unit cell.

Extended Characterization and Impact of Visible Fatty Acid Particles - A Case Study With a mAb Product.

In recent years, there has been increased scrutiny on the presence and formation of product-related particles in biopharmaceutical formulations. These types of particles, originating from the degradation of the active pharmaceutical ingredient or the excipients, can be challenging to identify and characterize due to their fragility. Additionally, the mechanisms of their formation as well as the impact of their presence on drug product safety can be complicated to elucidate. In this work, a case study is presented in which multiple batches of one formulated monoclonal antibody (mAb-A) were analyzed at different batch ages to better understand the formation of visible particles resulting from degradation of the surfactant polysorbate 20. The particle identity was determined by Raman spectroscopy as free fatty acid (FFA) and the particle composition over time was monitored by mass spectrometry. Further experimental work includes the counts and morphologies of subvisible particles by flow imaging microscopy. Finally, we evaluated the consequences of saline and human plasma exposure to the visible particles to better understand their fate upon dilution and/or administration which is routinely performed in the clinical setting. The experiments performed in this work can be used to support risk assessments of visible product-related particles.

Overexpression, isolation, and spectroscopic characterization of the bidirectional [NiFe] hydrogenase from Synechocystis sp. PCC 6803.

The bidirectional [NiFe] hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 was purified to apparent homogeneity by a single affinity chromatography step using a Synechocystis mutant with a Strep-tag II fused to the C terminus of HoxF. To increase the yield of purified enzyme and to test its overexpression capacity in Synechocystis the psbAII promoter was inserted upstream of the hoxE gene. In addition, the accessory genes (hypF, C, D, E, A, and B) from Nostoc sp. PCC 7120 were expressed under control of the psbAII promoter. The respective strains show higher hydrogenase activities compared with the wild type. For the first time a Fourier transform infrared (FTIR) spectroscopic characterization of a [NiFe] hydrogenase from an oxygenic phototroph is presented, revealing that two cyanides and one carbon monoxide coordinate the iron of the active site. At least four different redox states of the active site were detected during the reversible activation/inactivation. Although these states appear similar to those observed in standard [NiFe] hydrogenases, no paramagnetic nickel state could be detected in the fully oxidized and reduced forms. Electron paramagnetic resonance spectroscopy confirms the presence of several iron-sulfur clusters after reductive activation. One [4Fe4S](+) and at least one [2Fe2S](+) cluster could be identified. Catalytic amounts of NADH or NADPH are sufficient to activate the reaction of this enzyme with hydrogen.

Impact of amino acid substitutions near the catalytic site on the spectral properties of an O2-tolerant membrane-bound [NiFe] hydrogenase.

[NiFe] hydrogenases are widespread among microorganisms and catalyze the reversible cleavage of molecular hydrogen. However, only a few bacteria, such as Ralstonia eutropha H16 (Re), synthesize [NiFe] hydrogenases that perform H(2) cycling in the presence of O(2). These enzymes are of special interest for biotechnological applications. To gain further insight into the mechanism(s) responsible for the remarkable O(2) tolerance, we employ FTIR and EPR spectroscopy to study mutant variants of the membrane-bound hydrogenase (MBH) of Re-carrying substitutions of a particular cysteine residue in the vicinity of the [NiFe] active site that is characteristic of O(2)-tolerant membrane-bound [NiFe] hydrogenases. We demonstrate that these MBH variants, despite minor changes in the electronic structure and in the interaction behavior with the embedding protein matrix, display all relevant catalytic and noncatalytic states of the wild-type enzyme, as long as they are still located in the cytoplasmic membrane. Notably, in the oxidized Ni(r)-B state and the fully reduced forms, the CO stretching frequency increases with increasing polarity of the respective amino acid residue at the specific position of the cysteine residue. We purified the MBH mutant protein with a cysteine-to-alanine exchange to apparent homogeneity as dimeric enzyme after detergent solubilization from the membrane. This purified version displays increased oxygen sensitivity, which is reflected by detection of the oxygen-inhibited Ni(u)-A state, an irreversible inactive redox state, and the light-induced Ni(a)-L state even at room temperature.