Measuring the efficacy of standard and novel disinfection methods on frequently used physical therapy equipment: a 2-phase prospective randomized controlled trial.

BACKGROUND: Frequently used physical therapy (PT) equipment is difficult to disinfect due to equipment material and shape. The efficacy of standard disinfection of PT equipment is poorly understood. METHODS: We completed a 2-phase prospective microbiological analysis of fomites used in PT at our hospital from September 2022 to October 2023. For both phases, study fomites were obtained after usage and split into symmetrical halves for sampling. In phase 1, sides were sampled following standard disinfection. In phase 2, sides were randomized 1:1 to intervention or control. Samples were obtained before and after the intervention, a disinfection cabinet using Ultraviolet C (UV-C) and 6% nebulized hydrogen peroxide. We defined antimicrobial-resistant clinically important pathogens (AMR CIP) as methicillin-resistant staphylococcus aureus (MRSA), Vancomycin Resistant Enterococcus (VRE), and Multidrug resistant (MDR)-Gram-negatives and non-AMR CIP as methicillin-sensitive staphylococcus aureus (MSSA), Vancomycin sensitive Enterococcus (VSE), and Gram-negatives. Three assessments were made: 1) contamination following standard disinfection (phase 1), 2) contamination postintervention compared to no disinfection (phase 2) and, 3) contamination following standard disinfection compared to postintervention (phase 1 vs phase 2 intervention). RESULTS: The median total colony-forming units (CFU) from 122 study fomite samples was 1,348 (IQR 398-2,365). At the sample level, 52(43%) and 15(12%) of samples harbored any clinically important pathogens (CIPs) or AMR CIPs, respectively. The median CFU was 0 (IQR 0-55) in the intervention group and 977 (409-2,547) in the control group (P < .00001). CONCLUSION: Following standard disinfection, PT equipment remained heavily contaminated including AMR and non-AMR CIPs. Following the intervention, PT equipment was less contaminated and harbored no AMR CIPs compared to control sides supporting the efficacy of the intervention on difficult-to-disinfect PT fomites.

Mycobacterium immunogenum acquisition from hospital tap water: a genomic and epidemiologic analysis.

We identified 23 cases of Mycobacterium immunogenum respiratory acquisition linked to a colonized plumbing system at a new hospital addition. We conducted a genomic and epidemiologic investigation to assess for clonal acquisition of M. immunogenum from hospital water sources and improve understanding of genetic distances between M. immunogenum isolates. We performed whole-genome sequencing on 28 M. immunogenum isolates obtained from August 2013 to July 2021 from patients and water sources on four intensive care and intermediate units at an academic hospital. Study hospital isolates were recovered from 23 patients who experienced de novo respiratory isolation of M. immunogenum and from biofilms obtained from five tap water outlets. We also analyzed 10 M. immunogenum genomes from previously sequenced clinical (n = 7) and environmental (n = 3) external control isolates. The 38-isolate cohort clustered into three clades with pairwise single-nucleotide polymorphism (SNP) distances ranging from 0 to 106,697 SNPs. We identified two clusters of study hospital isolates in Clade 1 and one cluster in Clade 2 for which clinical and environmental isolates differed by fewer than 10 SNPs and had less than 0.5% accessory genome variation. A less restrictive combined threshold of 40 SNPs and 5% accessory genes reliably captured additional isolates that met clinical criteria for hospital acquisition, but 12 (4%) of 310 epidemiologically unrelated isolate pairs also met this threshold. Core and accessory genome analyses confirmed respiratory acquisition of multiple clones of M. immunogenum from hospital water sources to patients. When combined with epidemiologic investigation, genomic thresholds accurately distinguished hospital acquisition.

Comparative analysis of sampling and detection methods for fungal contamination on common healthcare environment surface materials.

We evaluated sampling and detection methods for fungal contamination on healthcare surface materials, comparing the efficacy of foam sponges, flocked swabs, and Replicate Organism Detection And Counting (RODAC) plates alongside culture-based quantification and quantitative polymerase chain reaction (qPCR). Findings indicate that sponge sampling and qPCR detection performed best, suggesting a foundation for future studies aiming to surveillance practices for fungi.

Healthcare Provider N95 Respirator Contamination Worn Behind Face Shields With SARS-CoV-2 During Routine Clinical Care of Patients With COVID-19.

N95 respirator contamination with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during clinical care of patients with coronavirus disease 2019 is poorly understood. We performed a prospective observational study on healthcare provider's (HCP's) N95 respirators' and face shields' SARS-CoV-2 contamination during aerosol-generating procedures on SARS-CoV-2-positive patients housed in a COVID-19-specific unit. Medical masks worn on top of HCP's N95 respirators, and under face shields, during study aerosol-generating procedures were used as surrogates to detect contamination to avoid waste. Thirty-three HCPs were studied, and a total of 33 mask and 27 face shields were sampled. Masks were cut into 9 pieces and face shields were sampled twice, front and back, to determine locality of contamination; however, no positive samples were identified using standard polymerase chain reaction techniques with a CT value up to 40. All 9 mask piece samples were then pooled, as were face shield samples, using centrifugal concentration with polyethersulfone membranes. Once pooled and concentrated, overall, 9 (15%) samples were positive via real-time polymerase chain reaction: 5 from masks (15.2%) and 4 from face shields (14.8%).

Identification of carbapenem-resistant organism (CRO) contamination of in-room sinks in intensive care units in a new hospital bed tower.

BACKGROUND: The origins and timing of inpatient room sink contamination with carbapenem-resistant organisms (CROs) are poorly understood. METHODS: We performed a prospective observational study to describe the timing, rate, and frequency of CRO contamination of in-room handwashing sinks in 2 intensive care units (ICU) in a newly constructed hospital bed tower. Study units, A and B, were opened to patient care in succession. The patients in unit A were moved to a new unit in the same bed tower, unit B. Each unit was similarly designed with 26 rooms and in-room sinks. Microbiological samples were taken every 4 weeks from 3 locations from each study sink: the top of the bowl, the drain cover, and the p-trap. The primary outcome was sink conversion events (SCEs), defined as CRO contamination of a sink in which CRO had not previously been detected. RESULTS: Sink samples were obtained 22 times from September 2020 to June 2022, giving 1,638 total environmental cultures. In total, 2,814 patients were admitted to study units while sink sampling occurred. We observed 35 SCEs (73%) overall; 9 sinks (41%) in unit A became contaminated with CRO by month 10, and all 26 sinks became contaminated in unit B by month 7. Overall, 299 CRO isolates were recovered; the most common species were Enterobacter cloacae and Pseudomonas aeruginosa. CONCLUSION: CRO contamination of sinks in 2 newly constructed ICUs was rapid and cumulative. Our findings support in-room sinks as reservoirs of CRO and emphasize the need for prevention strategies to mitigate contamination of hands and surfaces from CRO-colonized sinks.

Racial disparities in Clostridioides difficile testing in three southeastern US hospitals.

OBJECTIVE: To analyze Clostridioides difficile testing in 3 hospitals in central North Carolina to validate previous racial health-disparity findings. METHODS: We completed a retrospective analysis of inpatient C. difficile tests from 2015 to 2021 at 3 university-affiliated hospitals in North Carolina. We calculated the number of C. difficile tests per 1,000 patient days stratified by race: White, Black, and non-White, non-Black (NWNB). We defined a unique C. difficile test as one that occurred in an inpatient unit with a matching laboratory accession ID and on differing calendar days. Tests were evaluated overall, by hospital, by year, and by positivity rate. RESULTS: In total, 35,160 C. difficile tests and 2,571,850 patient days across all 3 hospitals from 2015 to 2021 were analyzed. The median number of C. difficile tests per 1,000 patient days was 13.85 (interquartile range [IQR], 9.88-16.07). Among all C. difficile tests, 5,225 (15%) were positive. White patients were administered more C. difficile tests (14.46 per 1,000 patient days) than Black patients (12.96; P < .0001) or NWNB race patients (10.27; P < .0001). Black patients were administered more tests than NWNB patients (P < .0001). White patients tested positive at a similar rate to Black patients (15% vs 15%; P = .3655) and higher than NWNB individuals (12%; P = .0061), and Black patients tested positive at a higher rate than NWNB patients (P = .0024). CONCLUSION: White patients received more C. difficile tests than Black and NWNB patient groups when controlling for race patient days. Future studies should control for comorbidities and investigate community onset of C. difficile by race and ethnicity.

A cluster of three extrapulmonary Mycobacterium abscessus infections linked to well-maintained water-based heater-cooler devices.

BACKGROUND: Various water-based heater-cooler devices (HCDs) have been implicated in nontuberculous mycobacteria outbreaks. Ongoing rigorous surveillance for healthcare-associated M. abscessus (HA-Mab) put in place following a prior institutional outbreak of M. abscessus alerted investigators to a cluster of 3 extrapulmonary M. abscessus infections among patients who had undergone cardiothoracic surgery. METHODS: Investigators convened a multidisciplinary team and launched a comprehensive investigation to identify potential sources of M. abscessus in the healthcare setting. Adherence to tap water avoidance protocols during patient care and HCD cleaning, disinfection, and maintenance practices were reviewed. Relevant environmental samples were obtained. Patient and environmental M. abscessus isolates were compared using multilocus-sequence typing and pulsed-field gel electrophoresis. Smoke testing was performed to evaluate the potential for aerosol generation and dispersion during HCD use. The entire HCD fleet was replaced to mitigate continued transmission. RESULTS: Clinical presentations of case patients and epidemiologic data supported intraoperative acquisition. M. abscessus was isolated from HCDs used on patients and molecular comparison with patient isolates demonstrated clonality. Smoke testing simulated aerosolization of M. abscessus from HCDs during device operation. Because the HCD fleet was replaced, no additional extrapulmonary HA-Mab infections due to the unique clone identified in this cluster have been detected. CONCLUSIONS: Despite adhering to HCD cleaning and disinfection strategies beyond manufacturer instructions for use, HCDs became colonized with and ultimately transmitted M. abscessus to 3 patients. Design modifications to better contain aerosols or filter exhaust during device operation are needed to prevent NTM transmission events from water-based HCDs.

Disinfection efficacy of Oxivir TB wipe residue on severe acute respiratory coronavirus virus 2 (SARS-CoV-2).

We assessed Oxivir Tb wipe disinfectant residue in a controlled laboratory setting to evaluate low environmental contamination of SARS-CoV-2. Frequency of viral RNA detection was not statistically different between intervention and control arms on day 3 (P=0.14). Environmental contamination viability is low; residual disinfectant did not significantly contribute to low contamination.

Second-sphere tuning of analogues for the ferric-hydroperoxoheme form of Mycobacterium tuberculosis MhuD.

Mycobacterium tuberculosis MhuD catalyzes the oxygenation of heme to mycobilin; experimental data presented here elucidates the novel hydroxylation reaction catalyzed by this enzyme. Analogues for the critical ferric-hydroperoxoheme (MhuD-heme-OOH) intermediate of this enzyme were characterized using UV/Vis absorption (Abs), circular dichroism (CD), and magnetic CD (MCD) spectroscopies. In order to extract electronic transition energies from these spectroscopic data, a novel global fitting model was developed for analysis of UV/Vis Abs, CD, and MCD data. A variant of MhuD was prepared, N7S, which weakens the affinity of heme-bound enzyme for a hydroperoxo analogue, azide, without significantly altering the protein secondary structure. Global fitting of spectroscopic data acquired in this study revealed that the second-sphere N7S substitution perturbs the electronic structure of two analogues for MhuD-heme-OOH: azide-inhibited MhuD (MhuD-heme-N3) and cyanide-inhibited MhuD (MhuD-heme-CN). The ground state electronic structures of MhuD-heme-N3 and MhuD-heme-CN were assessed using variable-temperature, variable-field MCD. Altogether, these data strongly suggest that there is a hydrogen bond between the Asn7 side-chain and the terminal oxygen of the hydroperoxo ligand in MhuD-heme-OOH. As discussed herein, this finding supports a novel hydroxylation reaction mechanism where the Asn7 side-chain guides a transient hydroxyl radical derived from homolysis of the OO bond in MhuD-heme-OOH to the ß- or δ-meso carbon of the porphyrin ligand yielding ß- or δ-meso-hydroxyheme, respectively.

An Enhanced Strategy for Daily Disinfection in Acute Care Hospital Rooms: A Randomized Clinical Trial.

Importance: Environmental contamination is a source of transmission between patients, health care practitioners, and other stakeholders in the acute care setting. Objective: To compare the efficacy of an enhanced daily disinfection strategy vs standard disinfection in acute care hospital rooms. Design, Setting, and Participants: This randomized clinical trial (RCT) was conducted in acute care hospital rooms at Duke University Hospital in Durham, North Carolina, from November 2021 to March 2022. Rooms were occupied by patients with contact precautions. Room surfaces (bed rails, overbed table, and in-room sink) were divided into 2 sides (right vs left), allowing each room to serve as its own control. Each side was randomized 1:1 to the intervention group or control group. Interventions: The intervention was a quaternary ammonium, salt-based, 24-hour continuously active germicidal wipe. It was applied in addition to routine disinfection for the intervention group. The control group received no intervention beyond routine disinfection. Main Outcomes and Measures: The primary outcome was the total contamination, measured in colony-forming units (CFUs) on the bed rails, overbed table, and sink on study day 1. The secondary outcomes were the proportion of sample areas with positive test results for clinically important pathogens, including methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus, and carbapenem-resistant Enterobacteriaceae; the similarity in baseline contamination between sample area sides on study day 0 before application of the intervention, and the proportion of sample areas with removed UV luminescent gel on study day 1. Results: A total of 50 study rooms occupied by 50 unique patients (median [IQR] age, 61 [45-69] years; 26 men [52%]) with contact precautions were enrolled. Of these patients, 41 (82%) were actively receiving antibiotics, 39 (78%) were bedridden, and 28 (56%) had active infections with study-defined clinically important pathogens. On study day 1, the median (IQR) total CFUs for the intervention group was lower than that for the control group (3561 [1292-7602] CFUs vs 5219 [1540-12 364] CFUs; P = .002). On study day 1, the intervention side was less frequently contaminated with patient-associated clinically important pathogens compared with the control side of the room (4 [14%] vs 11 [39%]; P = .04). Conclusions and Relevance: Results of this RCT demonstrated that a quaternary ammonium, salt-based, 24-hour continuously active germicidal wipe decreased the environmental bioburden in acute care hospital rooms compared with routine disinfection. The findings warrant large-scale RCTs to determine whether enhanced daily disinfection strategies can decrease patient acquisition and adverse patient outcomes. Trial Registration: ClinicalTrials.gov Identifier: NCT05560321.

The Impact of Infection Versus Colonization on Clostridioides difficile Environmental Contamination in Hospitalized Patients With Diarrhea.

Background: Patients with Clostridioides difficile infections (CDIs) contaminate the healthcare environment; however, the relative contribution of contamination by colonized individuals is unknown. Current guidelines do not recommend the use of contact precautions for asymptomatic C difficile carriers. We evaluated C difficile environmental contamination in rooms housing adult inpatients with diarrhea based on C difficile status. Methods: We performed a prospective cohort study of inpatient adults with diarrhea who underwent testing for CDI via polymerase chain reaction (PCR) and enzyme immunoassay (EIA). Patients were stratified into cohorts based on test result: infected (PCR+/EIA+), colonized (PCR+/EIA-), or negative/control (PCR-). Environmental microbiological samples were taken within 24 hours of C difficile testing and again for 2 successive days. Samples were obtained from the patient, bathroom, and care areas. Results: We enrolled 94 patients between November 2019 and June 2021. Clostridioides difficile was recovered in 93 (38%) patient rooms: 44 (62%) infected patient rooms, 35 (43%) colonized patient rooms (P = .08 vs infected 38 patient rooms), and 14 (15%) negative patient rooms (P < .01 vs infected; P < .01 vs colonized). Clostridioides difficile was recovered in 40 (56%), 6 (9%), and 20 (28%) of bathrooms, care areas and patient areas in 40 infected patient rooms; 34 (41%), 1 (1%), and 4 (5%) samples in colonized patient rooms; and 12 (13%), 1 (1%), and 3 (3%) of samples in negative patient rooms, respectively. Conclusions: Patients colonized with C difficile frequently contaminated the hospital environment. Our data support the use of contact precautions when entering rooms of patients colonized with C difficile, especially when entering the bathroom.

Severe acute respiratory syndrome coronavirus 2 environmental contamination in hospital rooms is uncommon using viral culture techniques.

We assessed environmental contamination of inpatient rooms housing coronavirus disease 2019 (COVID-19) patients in a dedicated COVID-19 unit. Contamination with severe acute respiratory syndrome coronavirus 2 was found on 5.5% (19/347) of surfaces via reverse transcriptase polymerase chain reaction and 0.3% (1/347) of surfaces via cell culture. Environmental contamination is uncommon in hospitals rooms; RNA presence is not a specific indicator of infectious virus.

A Dynamic Substrate is Required for MhuD-Catalyzed Degradation of Heme to Mycobilin.

The noncanonical heme oxygenase MhuD from Mycobacterium tuberculosis binds a heme substrate that adopts a dynamic equilibrium between planar and out-of-plane ruffled conformations. MhuD degrades this substrate to an unusual mycobilin product via successive monooxygenation and dioxygenation reactions. This article establishes a causal relationship between heme substrate dynamics and MhuD-catalyzed heme degradation, resulting in a refined enzymatic mechanism. UV/vis absorption (Abs) and electrospray ionization mass spectrometry (ESI-MS) data demonstrated that a second-sphere substitution favoring the population of the ruffled heme conformation changed the rate-limiting step of the reaction, resulting in a measurable buildup of the monooxygenated meso-hydroxyheme intermediate. In addition, UV/vis Abs and ESI-MS data for a second-sphere variant that favored the planar substrate conformation showed that this change altered the enzymatic mechanism resulting in an α-biliverdin product. Single-turnover kinetic analyses for three MhuD variants revealed that the rate of heme monooxygenation depends upon the population of the ruffled substrate conformation. These kinetic analyses also revealed that the rate of meso-hydroxyheme dioxygenation by MhuD depends upon the population of the planar substrate conformation. Thus, the ruffled heme conformation supports rapid heme monooxygenation by MhuD, but further oxygenation to the mycobilin product is inhibited. In contrast, the planar substrate conformation exhibits altered heme monooxygenation regiospecificity followed by rapid oxygenation of meso-hydroxyheme. Altogether, these data yielded a refined enzymatic mechanism for MhuD where access to both substrate conformations is needed for rapid incorporation of three oxygen atoms into heme yielding mycobilin.

The affinity of MhuD for heme is consistent with a heme degrading function in vivo.

MhuD is a protein found in mycobacteria that can bind up to two heme molecules per protein monomer and catalyze the degradation of heme to mycobilin in vitro. Here the Kd1 for heme dissociation from heme-bound MhuD was determined to be 7.6 ± 0.8 nM and the Kd2 for heme dissocation from diheme-bound MhuD was determined to be 3.3 ± 1.1 µM. These data strongly suggest that MhuD is a competent heme oxygenase in vivo.

Selective Small Molecule Recognition of RNA Base Pairs.

Many types of RNAs exist in the human transcriptome, yet only the bacterial ribosome has been exploited as a small molecule drug target. Aside from rRNA, other cellular RNAs such as noncoding RNAs have primarily secondary structure and limited tertiary structure. Within these secondary structures of noncanonically paired and unpaired regions, more than 50% are base paired, with most efforts to target these structures focused on looped regions. A void exists in the availability of small molecules capable of targeting RNA base pairs. Using chemoinformatics, an RNA-focused library enriched for nitrogen-containing heterocycles was developed and tested for binding RNA base pairs, leading to the identification of six selective and previously unknown binders. While all binders were derivatives of benzimidazoles, those with expanded aromatic polycycles bound selectively to AU pairs, while those with flexible urea side chains bound selectively to GC pairs. Two of the three selective GC pair binders can distinguish between two different orientations, 5'GG/3'CC and 5'GC/3'CG pairs. Furthermore, all six molecules showed >50-fold selectivity for RNA over DNA. These studies provide foundational knowledge to better exploit RNA as targets for small molecule chemical probes or lead therapeutics by using modules that target RNA base pairs.

Correction: Dynamic ruffling distortion of the heme substrate in non-canonical heme oxygenase enzymes.

Correction for 'Dynamic ruffling distortion of the heme substrate in non-canonical heme oxygenase enzymes' by Amanda B. Graves et al., Dalton Trans., 2016, 45, 10058-10067.

Dynamic ruffling distortion of the heme substrate in non-canonical heme oxygenase enzymes.

Recent work by several groups has established that MhuD, IsdG, and IsdI are non-canonical heme oxygenases that induce significant out-of-plane ruffling distortions of their heme substrates enroute to mycobilin or staphylobilin formation. However, clear explanations for the observations of "nested" S = ½ VTVH MCD saturation magnetization curves at cryogenic temperatures, and exchange broadened (1)H NMR resonances at physiologically-relevant temperatures have remained elusive. Here, MCD and NMR data have been acquired for F23A and F23W MhuD-heme-CN, in addition to MCD data for IsdI-heme-CN, in order to complete assembly of a library of spectroscopic data for cyanide-inhibited ferric heme with a wide range of ruffling deformations. The spectroscopic data were used to evaluate a number of computational models for cyanide-inhibited ferric heme, which ultimately led to the development of an accurate NEVPT2/CASSCF model. The resulting model has a shallow, double-well potential along the porphyrin ruffling coordinate, which provides clear explanations for the unusual MCD and NMR data. The shallow, double-well potential also implies that MhuD-, IsdG-, and IsdI-bound heme is dynamic, and the functional implications of these dynamics are discussed.

Measurement of Heme Ruffling Changes in MhuD Using UV-vis Spectroscopy.

For decades it has been known that an out-of-plane ruffling distortion of heme perturbs its UV-vis absorption (Abs) spectrum, but whether increased ruffling induces a red or blue shift of the Soret band has remained a topic of debate. This debate has been resolved by the spectroscopic and computational characterization of Mycobacterium tuberculosis MhuD presented here, an enzyme that converts heme, oxygen, and reducing equivalents to nonheme iron and mycobilin. W66F and W66A MhuD have been characterized using (1)H nuclear magnetic resonance, Abs, and magnetic circular dichroism spectroscopies, and the data have been used to develop an experimentally validated theoretical model of ruffled, ferric heme. The PBE density functional theory (DFT) model that has been developed accurately reproduces the observed spectral changes from wild type enzyme, and the underlying quantum mechanical origins of these ruffling-induced changes were revealed by analyzing the PBE DFT description of the electronic structure. Small amounts of heme ruffling have no influence on the energy of the Q-band and blue-shift the Soret band due to symmetry-allowed mixing of the Fe 3dxy and porphyrin a2u orbitals. Larger amounts of ruffling red-shift both the Q and Soret bands due to disruption of π-bonding within the porphyrin ring.

Bioactivity of a Rice Bran-Derived Peptide and its Sensory Evaluation and Storage Stability in Orange Juice.

A pentapeptide prepared from rice bran demonstrated growth inhibition on human lung, liver, breast, and colon cancer cell lines. The objectives of this study were to evaluate the human prostate cancer growth inhibition by the pentapeptide and its 6-mo storage stability by incorporating spray-dried orange juice, and determining sensory acceptability. The pentapeptide showed inhibition of human prostate cancer cells by 45% at 460 µg/mL concentration. When incorporated in spray-dried orange juice, and reconstituted with water and tested, there was an approximately 10% degradation of the peptide at 620 µg/mL concentration under refrigerated conditions over a 6 mo storage period, whereas at ambient temperature the degradation was 30%. Larger degradation was observed when 240 or 460 µg/mL pentapeptide was used. Overall, consumer panelists liked sensory aspect of the reconstituted pentapeptide incorporated orange juice beverage. Also consumer panelists liked the color and mouthfeel attributes, their hedonic impression of flavor attribute was slightly low due to unpalatable bitter note caused by the presence of the peptide. Incorporation of the pentapeptide in spray-dried orange juice has the potential to serve as a functional food ingredient that can offer health benefits to consumers. It is possible that the structural instability can be minimized by encapsulation.

Crystallographic and spectroscopic insights into heme degradation by Mycobacterium tuberculosis MhuD.

Mycobacterium heme utilization degrader (MhuD) is a heme-degrading protein from Mycobacterium tuberculosis responsible for extracting the essential nutrient iron from host-derived heme. MhuD has been previously shown to produce unique organic products compared to those of canonical heme oxygenases (HOs) as well as those of the IsdG/I heme-degrading enzymes from Staphylococcus aureus. Here, we report the X-ray crystal structure of cyanide-inhibited MhuD (MhuD-heme-CN) as well as detailed (1)H nuclear magnetic resonance (NMR), UV/vis absorption, and magnetic circular dichroism (MCD) spectroscopic characterization of this species. There is no evidence for an ordered network of water molecules on the distal side of the heme substrate in the X-ray crystal structure, as was previously reported for canonical HOs. The degree of heme ruffling in the crystal structure of MhuD is greater than that observed for HO and less than that observed for IsdI. As a consequence, the Fe 3dxz-, 3dyz-, and 3dxy-based MOs are very close in energy, and the room-temperature (1)H NMR spectrum of MhuD-heme-CN is consistent with population of both a (2)Eg electronic state with a (dxy)(2)(dxz,dyz)(3) electron configuration, similar to the ground state of canonical HOs, and a (2)B2g state with a (dxz,dyz)(4)(dxy)(1) electron configuration, similar to the ground state of cyanide-inhibited IsdI. Variable temperature, variable field MCD saturation magnetization data establishes that MhuD-heme-CN has a (2)B2g electronic ground state with a low-lying (2)Eg excited state. Our crystallographic and spectroscopic data suggest that there are both structural and electronic contributions to the α-meso regioselectivity of MhuD-catalyzed heme cleavage. The structural distortion of the heme substrate observed in the X-ray crystal structure of MhuD-heme-CN is likely to favor cleavage at the α- and γ-meso carbons, whereas the spin density distribution may favor selective oxygenation of the α-meso carbon.