Functional maturation of cytochromes P450 3A4 and 2D6 relies on GAPDH- and Hsp90-Dependent heme allocation.

Cytochrome P450 3A4 and 2D6 (EC 1.14.13.97 and 1.14.14.1; CYP3A4 and 2D6) are heme-containing enzymes that catalyze the oxidation of a wide number of xenobiotic and drug substrates and thus broadly impact human biology and pharmacologic therapies. Although their activities are directly proportional to their heme contents, little is known about the cellular heme delivery and insertion processes that enable their maturation to functional form. We investigated the potential involvement of GAPDH and chaperone Hsp90, based on our previous studies linking these proteins to intracellular heme allocation. We studied heme delivery and insertion into CYP3A4 and 2D6 after they were transiently expressed in HEK293T and GlyA CHO cells or when naturally expressed in HEPG2 cells in response to rifampicin, and also investigated their associations with GAPDH and Hsp90 in cells. The results indicate that GAPDH and its heme binding function is involved in delivery of mitochondria-generated heme to apo-CYP3A4 and 2D6, and that cell chaperone Hsp90 is additionally involved in driving their heme insertions. Uncovering how cells allocate heme to CYP3A4 and 2D6 provides new insight on their maturation processes and how this may help to regulate their functions in health and disease.

Indoleamine dioxygenase and tryptophan dioxygenase activities are regulated through control of cell heme allocation by nitric oxide.

Indoleamine-2, 3-dioxygenase (IDO1) and Tryptophan-2, 3-dioxygenase (TDO) catalyze the conversion of L-tryptophan to N-formyl-kynurenine and thus play primary roles in metabolism, inflammation, and tumor immune surveillance. Because their activities depend on their heme contents, which vary in biological settings and go up or down in a dynamic manner, we studied how their heme levels may be impacted by nitric oxide (NO) in mammalian cells. We utilized cells expressing TDO or IDO1 either naturally or via transfection and determined their activities, heme contents, and expression levels as a function of NO exposure. We found NO has a bimodal effect: a narrow range of low NO exposure promoted cells to allocate heme into the heme-free TDO and IDO1 populations and consequently boosted their heme contents and activities 4- to 6-fold, while beyond this range the NO exposure transitioned to have a negative impact on their heme contents and activities. NO did not alter dioxygenase protein expression levels, and its bimodal impact was observed when NO was released by a chemical donor or was generated naturally by immune-stimulated macrophage cells. NO-driven heme allocations to IDO1 and TDO required participation of a GAPDH-heme complex and for IDO1 required chaperone Hsp90 activity. Thus, cells can up- or downregulate their IDO1 and TDO activities through a bimodal control of heme allocation by NO. This mechanism has important biomedical implications and helps explain why the IDO1 and TDO activities in animals go up and down in response to immune stimulation.

Biochemical, Biophysical, and Structural Analysis of an Unusual DyP from the Extremophile Deinococcus radiodurans.

Dye-decolorizing peroxidases (DyPs) are heme proteins with distinct structural properties and substrate specificities compared to classical peroxidases. Here, we demonstrate that DyP from the extremely radiation-resistant bacterium Deinococcus radiodurans is, like some other homologues, inactive at physiological pH. Resonance Raman (RR) spectroscopy confirms that the heme is in a six-coordinated-low-spin (6cLS) state at pH 7.5 and is thus unable to bind hydrogen peroxide. At pH 4.0, the RR spectra of the enzyme reveal the co-existence of high-spin and low-spin heme states, which corroborates catalytic activity towards H2O2 detected at lower pH. A sequence alignment with other DyPs reveals that DrDyP possesses a Methionine residue in position five in the highly conserved GXXDG motif. To analyze whether the presence of the Methionine is responsible for the lack of activity at high pH, this residue is substituted with a Glycine. UV-vis and RR spectroscopies reveal that the resulting DrDyPM190G is also in a 6cLS spin state at pH 7.5, and thus the Methionine does not affect the activity of the protein. The crystal structures of DrDyP and DrDyPM190G, determined to 2.20 and 1.53 Å resolution, respectively, nevertheless reveal interesting insights. The high-resolution structure of DrDyPM190G, obtained at pH 8.5, shows that one hydroxyl group and one water molecule are within hydrogen bonding distance to the heme and the catalytic Asparagine and Arginine. This strong ligand most likely prevents the binding of the H2O2 substrate, reinforcing questions about physiological substrates of this and other DyPs, and about the possible events that can trigger the removal of the hydroxyl group conferring catalytic activity to DrDyP.

O2 Carrier Myoglobin Also Exhibits ß-Lactamase Activity That Is Regulated by the Heme Coordination State.

Heme proteins perform a variety of biological functions and also play significant roles in the field of bio-catalysis. The ß-lactamase activity of heme proteins has rarely been reported. Herein, we found, for the first time, that myoglobin (Mb), an O2 carrier, also exhibits novel ß-lactamase activity by catalyzing the hydrolysis of ampicillin. The catalytic proficiency ((kcat/KM)/kuncat) was determined to be 6.25 × 1010, which is much higher than the proficiency reported for designed metalloenzymes, although it is lower than that of natural ß-lactamases. Moreover, we found that this activity could be regulated by an engineered disulfide bond, such as Cys46-Cys61 in F46C/L61C Mb or by the addition of imidazole to directly coordinate to the heme center. These results indicate that the heme active site is responsible for the ß-lactamase activity of Mb. Therefore, the study suggests the potential of heme proteins acting as ß-lactamases, which broadens the diversity of their catalytic functions.

Heme-Protein Interactions and Functional Relevant Heme Deformations: The Cytochrome c Case.

Heme proteins are known to perform a plethora of biologically important functions. This article reviews work that has been conducted on various class I cytochrome c proteins over a period of nearly 50 years. The article focuses on the relevance of symmetry-lowering heme-protein interactions that affect the function of the electron transfer protein cytochrome c. The article provides an overview of various, mostly spectroscopic studies that explored the electronic structure of the heme group in these proteins and how it is affected by symmetry-lowering deformations. In addition to discussing a large variety of spectroscopic studies, the article provides a theoretical framework that should enable a comprehensive understanding of the physical chemistry that underlies the function not only of cytochrome c but of all heme proteins.

Chemical binding of horseradish peroxidase enzyme with poly beta-cyclodextrin and its application as molecularly imprinted polymer for the monitoring of H2 O2 in human plasma samples.

In this study, a selective and sensitive molecular imprinting-based electrochemical sensors, for horseradish peroxidase (HRP) entrapment was fabricated using electro polymerization of ß-Cyclodextrin (ß-CD) on the surface of glassy carbon electrode. Poly beta-cyclodextrin P(ß-CD) provide efficient surface area for self-immobilization of HRP as well as improve imprinting efficiency. The proposed imprinted biosensor successfully utilized for detection of HRP with excellent analytical results which linear range is 0.1 mg/mL to 10 ng/mL with LOD of 2.23 ng/mL. Furthermore, electrocatalytical activity of the prepared biosensor toward the reduction of hydrogen peroxide was investigated in the ranges of 1 to 15 µM with a detection limit of 0.4 µM by using chronoamperometry technique. The developed biosensor was used for the detection of hydrogen peroxide in unprocessed human plasma sample.

The pivotal role of heme Oxygenase-1 in reversing the pathophysiology and systemic complications of NAFLD.

The pathogenesis and molecular pathways involved in non-alcoholic fatty liver disease (NAFLD) are reviewed, as well as what is known about mitochondrial dysfunction that leads to heart disease and the progression to steatohepatitis and hepatic fibrosis. We focused our discussion on the role of the antioxidant gene heme oxygenase-1 (HO-1) and its nuclear coactivator, peroxisome proliferator-activated receptor-gamma coactivator (PGC1-α) in the regulation of mitochondrial biogenesis and function and potential therapeutic benefit for cardiac disease, NAFLD as well as the pharmacological effect they have on the chronic inflammatory state of obesity. The result is increased mitochondrial function and the conversion of white adipocyte tissue to beige adipose tissue ("browning of white adipose tissue") that leads to an improvement in signaling pathways and overall liver function. Improved mitochondrial biogenesis and function is essential to preventing the progression of hepatic steatosis to NASH and cirrhosis as well as preventing cardiovascular complications.

Noncanonical Heme Ligands Steer Carbene Transfer Reactivity in an Artificial Metalloenzyme*.

Changing the primary metal coordination sphere is a powerful strategy for tuning metalloprotein properties. Here we used amber stop codon suppression with engineered pyrrolysyl-tRNA synthetases, including two newly evolved enzymes, to replace the proximal histidine in myoglobin with Nδ -methylhistidine, 5-thiazoylalanine, 4-thiazoylalanine and 3-(3-thienyl)alanine. In addition to tuning the heme redox potential over a >200 mV range, these noncanonical ligands modulate the protein's carbene transfer activity with ethyl diazoacetate. Variants with increased reduction potential proved superior for cyclopropanation and N-H insertion, whereas variants with reduced Eo values gave higher S-H insertion activity. Given the functional importance of histidine in many enzymes, these genetically encoded analogues could be valuable tools for probing mechanism and enabling new chemistries.

Myoglobin induces mitochondrial fusion, thereby inhibiting breast cancer cell proliferation.

Myoglobin is a monomeric heme protein expressed ubiquitously in skeletal and cardiac muscle and is traditionally considered to function as an oxygen reservoir for mitochondria during hypoxia. It is now well established that low concentrations of myoglobin are aberrantly expressed in a significant proportion of breast cancer tumors. Despite being expressed only at low levels in these tumors, myoglobin is associated with attenuated tumor growth and a better prognosis in breast cancer patients, but the mechanism of this myoglobin-mediated protection against further cancer growth remains unclear. Herein, we report a signaling pathway by which myoglobin regulates mitochondrial dynamics and thereby decreases cell proliferation. We demonstrate in vitro that expression of human myoglobin in MDA-MB-231, MDA-MB-468, and MCF7 breast cancer cells induces mitochondrial hyperfusion by up-regulating mitofusins 1 and 2, the predominant catalysts of mitochondrial fusion. This hyperfusion causes cell cycle arrest and subsequent inhibition of cell proliferation. Mechanistically, increased mitofusin expression was due to myoglobin-dependent free-radical production, leading to the oxidation and degradation of the E3 ubiquitin ligase parkin. We recapitulated this pathway in a murine model in which myoglobin-expressing xenografts exhibited decreased tumor volume with increased mitofusin, markers of cell cycle arrest, and decreased parkin expression. Furthermore, in human triple-negative breast tumor tissues, mitofusin and myoglobin levels were positively correlated. Collectively, these results elucidate a new function for myoglobin as a modulator of mitochondrial dynamics and reveal a novel pathway by which myoglobin decreases breast cancer cell proliferation and tumor growth by up-regulating mitofusin levels.

Mechanism and regulation of ferrous heme-nitric oxide (NO) oxidation in NO synthases.

Nitric oxide (NO) synthases (NOSs) catalyze the formation of NO from l-arginine. We have shown previously that the NOS enzyme catalytic cycle involves a large number of reactions but can be characterized by a global model with three main rate-limiting steps. These are the rate of heme reduction by the flavin domain (kr ), of dissociation of NO from the ferric heme-NO complex (kd ), and of oxidation of the ferrous heme-NO complex (kox). The reaction of oxygen with the ferrous heme-NO species is part of a futile cycle that does not directly contribute to NO synthesis but allows a population of inactive enzyme molecules to return to the catalytic cycle, and thus, enables a steady-state NO synthesis rate. Previously, we have reported that this reaction does involve the reaction of oxygen with the NO-bound ferrous heme complex, but the mechanistic details of the reaction, that could proceed via either an inner-sphere or an outer-sphere mechanism, remained unclear. Here, we present additional experiments with neuronal NOS (nNOS) and inducible NOS (iNOS) variants (nNOS W409F and iNOS K82A and V346I) and computational methods to study how changes in heme access and electronics affect the reaction. Our results support an inner-sphere mechanism and indicate that the particular heme-thiolate environment of the NOS enzymes can stabilize an N-bound FeIII-N(O)OO- intermediate species and thereby catalyze this reaction, which otherwise is not observed or favorable in proteins like globins that contain a histidine-coordinated heme.

Examination of abiotic cofactor assembly in photosynthetic biomimetics: site-specific stereoselectivity in the conjugation of a ruthenium(II) tris(bipyridine) photosensitizer to a multi-heme protein.

To understand design principles for assembling photosynthetic biohybrids that incorporate precisely-controlled sites for electron injection into redox enzyme cofactor arrays, we investigated the influence of chirality in assembly of the photosensitizer ruthenium(II)bis(2,2'-bipyridine)(4-bromomethyl-4'-methyl-2,2'-bipyridine), Ru(bpy)2(Br-bpy), when covalently conjugated to cysteine residues introduced by site-directed mutagenesis in the triheme periplasmic cytochrome A (PpcA) as a model biohybrid system. For two investigated conjugates that show ultrafast electron transfer, A23C-Ru and K29C-Ru, analysis by circular dichroism spectroscopy, CD, demonstrated site-specific chiral discrimination as a factor emerging from the close association between [Ru(bpy)3]2+ and heme cofactors. CD analysis showed the A23C-Ru and K29C-Ru conjugates to have distinct, but opposite, stereoselectivity for the Λ and Δ-Ru(bpy)2(Br-bpy) enantiomers, with enantiomeric excesses of 33.1% and 65.6%, respectively. In contrast, Ru(bpy)2(Br-bpy) conjugation to a protein site with high flexibility, represented by the E39C-Ru construct, exhibited a nearly negligible chiral selectivity, measured by an enantiomeric excess of 4.2% for the Λ enantiomer. Molecular dynamics simulations showed that site-specific stereoselectivity reflects steric constraints at the conjugating sites and that a high degree of chiral selectivity correlates to reduced structural disorder for [Ru(bpy)3]2+ in the linked assembly. This work identifies chiral discrimination as means to achieve site-specific, precise geometric positioning of introduced photosensitizers relative to the heme cofactors in manner that mimics the tuning of cofactors in photosynthesis.

A traffic light enzyme: acetate binding reversibly switches chlorite dismutase from a red- to a green-colored heme protein.

Chlorite dismutase is a unique heme enzyme that catalyzes the conversion of chlorite to chloride and molecular oxygen. The enzyme is highly specific for chlorite but has been known to bind several anionic and neutral ligands to the heme iron. In a pH study, the enzyme changed color from red to green in acetate buffer pH 5.0. The cause of this color change was uncovered using UV-visible and EPR spectroscopy. Chlorite dismutase in the presence of acetate showed a change of the UV-visible spectrum: a redshift and hyperchromicity of the Soret band from 391 to 404 nm and a blueshift of the charge transfer band CT1 from 647 to 626 nm. Equilibrium binding titrations with acetate resulted in a dissociation constant of circa 20 mM at pH 5.0 and 5.8. EPR spectroscopy showed that the acetate bound form of the enzyme remained high spin S = 5/2, however with an apparent change of the rhombicity and line broadening of the spectrum. Mutagenesis of the proximal arginine Arg183 to alanine resulted in the loss of the ability to bind acetate. Acetate was discovered as a novel ligand to chlorite dismutase, with evidence of direct binding to the heme iron. The green color is caused by a blueshift of the CT1 band that is characteristic of the high spin ferric state of the enzyme. Any weak field ligand that binds directly to the heme center may show the red to green color change, as was indeed the case for fluoride.

Construction of a new T7 promoter compatible Escherichia coli Nissle 1917 strain for recombinant production of heme-dependent proteins.

BACKGROUND: Heme proteins and heme-derived molecules are essential in numerous cellular processes. Research into their in vitro functionality requires the production of large amounts of protein. Unfortunately, high yield expression is hampered by the lack of E. coli strains naturally capable of taking up heme from the medium. We recently reported the use of the probiotic E. coli strain Nissle 1917 (EcN) to sufficiently produce heme containing proteins, as it encodes the outer membrane heme receptor, ChuA, which allows for natural uptake of heme. The EcN strain however lacks the gene for T7 RNA polymerase, which is necessary for the expression of genes under the control of the T7-promotor, widely used in expression vectors like the pET or pDuet series. RESULTS: A new T7-promoter compatible EcN strain was constructed by integrating the gene for T7-RNA polymerase under the control of a lacUV5 promoter into the malEFG operon of EcN. Test expressions of genes via T7 promoter-based vectors in the new EcN(T7) strain were successful. Expression in EcN(T7) resulted in the efficient production of recombinant heme proteins in which the heme cofactor was incorporated during protein production. In addition, the new EcN(T7) strain can be used to co-express genes for the production of heme-derived molecules like biliverdin or other linear tetrapyrroles. We demonstrate the successful recombinant production of the phytochromes BphP, from Pseudomonas aeruginosa, and Cph1, from Synechocystis sp. PCC6803, loaded with their linear tetrapyrrole cofactors, biliverdin and phycocyanobilin, respectively. CONCLUSION: We present a new E. coli strain for efficient production of heme proteins and heme-derived molecules using T7-promoter based expression vectors. The new EcN(T7) strain enables the use of a broader spectrum of expression vectors, as well as the co-expression of genes using the pDuet expression vectors, for expressing heme containing proteins. By utilizing E. coli strains EcN and EcN(T7), capable of being fed heme, the rate limiting step of heme biosynthesis in E. coli is eliminated, thereby permitting higher heme saturation of heme proteins and also higher yields of heme-derived molecules.

Mimochrome, a metalloporphyrin-based catalytic Swiss knife†.

Over the years, mimochromes, a class of miniaturized porphyrin-based metalloproteins, have proven to be reliable but still versatile scaffolds. After two decades from their birth, we retrospectively review our work in mimochrome design and engineering, which allowed us developing functional models. They act as electron-transfer miniproteins or more elaborate artificial metalloenzymes, endowed with peroxidase, peroxygenase, and hydrogenase activities. Mimochromes represent simple yet functional synthetic models that respond to metal ion replacement and noncovalent modulation of the environment, similarly to natural heme-proteins. More recently, we have demonstrated that the most active analogue retains its functionality when immobilized on nanomaterials and surfaces, thus affording bioconjugates, useful in sensing and catalysis. This review also briefly summarizes the most important contributions to heme-protein design from leading groups in the field.

Redox State Control of Human Cytoglobin by Direct Electrochemical Method to Investigate Its Function in Molecular Basis.

The direct electron transfer between human cytoglobin (Cygb) and the electrode surface, which would allow manipulating the oxidation states of the heme iron in Cygb, was first observed by immobilizing Cygb on a nanoporous gold (NPG) electrode via a carboxy-terminated alkanethiol. The voltammetric performances of the wild type and mutated Cygb-immobilized NPG electrodes were evaluated in the absence or presence of potential substrates. The obtained results demonstrated that the usefulness of the proposed method in understanding the function of Cygb in molecular basis.

NO Scavenging through Reductive Nitrosylation of Ferric Mycobacterium tuberculosis and Homo sapiens Nitrobindins.

Ferric nitrobindins (Nbs) selectively bind NO and catalyze the conversion of peroxynitrite to nitrate. In this study, we show that NO scavenging occurs through the reductive nitrosylation of ferric Mycobacterium tuberculosis and Homo sapiens nitrobindins (Mt-Nb(III) and Hs-Nb(III), respectively). The conversion of Mt-Nb(III) and Hs-Nb(III) to Mt-Nb(II)-NO and Hs-Nb(II)-NO, respectively, is a monophasic process, suggesting that over the explored NO concentration range (between 2.5 × 10-5 and 1.0 × 10-3 M), NO binding is lost in the mixing time (i.e., NOkon ≥ 1.0 × 106 M-1 s-1). The pseudo-first-order rate constant for the reductive nitrosylation of Mt-Nb(III) and Hs-Nb(III) (i.e., k) is not linearly dependent on the NO concentration but tends to level off, with a rate-limiting step (i.e., klim) whose values increase linearly with [OH-]. This indicates that the conversion of Mt-Nb(III) and Hs-Nb(III) to Mt-Nb(II)-NO and Hs-Nb(II)-NO, respectively, is limited by the OH--based catalysis. From the dependence of klim on [OH-], the values of the second-order rate constant kOH- for the reductive nitrosylation of Mt-Nb(III)-NO and Hs-Nb(III)-NO were obtained (4.9 (±0.5) × 103 M-1 s-1 and 6.9 (±0.8) × 103 M-1 s-1, respectively). This process leads to the inactivation of two NO molecules: one being converted to HNO2 and another being tightly bound to the ferrous heme-Fe(II) atom.

Gas Sensing by Bacterial H-NOX Proteins: An MD Study.

Gas sensing is crucial for both prokaryotes and eukaryotes and is primarily performed by heme-based sensors, including H-NOX domains. These systems may provide a new, alternative mode for transporting gaseous molecules in higher organisms, but for the development of such systems, a detailed understanding of the ligand-binding properties is required. Here, we focused on ligand migration within the protein matrix: we performed molecular dynamics simulations on three bacterial (Ka, Ns and Cs) H-NOX proteins and studied the kinetics of CO, NO and O2 diffusion. We compared the response of the protein structure to the presence of ligands, diffusion rate constants, tunnel systems and storage pockets. We found that the rate constant for diffusion decreases in the O2 > NO > CO order in all proteins, and in the Ns > Ks > Cs order if single-gas is considered. Competition between gases seems to seriously influence the residential time of ligands spent in the distal pocket. The channel system is profoundly determined by the overall fold, but the sidechain pattern has a significant role in blocking certain channels by hydrophobic interactions between bulky groups, cation-π interactions or hydrogen bonding triads. The majority of storage pockets are determined by local sidechain composition, although certain functional cavities, such as the distal and proximal pockets are found in all systems. A major guideline for the design of gas transport systems is the need to chemically bind the gas molecule to the protein, possibly joining several proteins with several heme groups together.

Correlation of indoleamine-2,3-dioxigenase 1 inhibitory activity of 4,6-disubstituted indazole derivatives and their heme binding affinity.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a heme-containing enzyme that acts on the first and rate-limiting step of the tryptophan/kynurenine pathway. Since the pathway is one of the means of cancer immune evasion, IDO1 inhibitors have drawn interest as potential therapeutics for cancers. We found a 4,6-disubstituted indazole 1 as a hit compound that showed both IDO1 inhibitory activity and binding affinity for IDO1 heme. Structural modification of 1 yielded compound 6, whose relatively large substituent at the 4-position and proper size substituent at the 6-position were found to be important for the enhancement of IDO1 inhibitory activity and heme affinity. A series of compounds synthesized in this work were evaluated by in silico docking simulations and by in vitro experiments using a C129Y mutant of the pocket-A of IDO1. Our results revealed that proper substituents at the 6- and 4-positions of the compounds interact with pockets A and B, respectively, and that, in particular, a good fit in pocket-A is important for the compounds' biological activities. Absorption spectral analysis of these compounds showed that they strongly bound to the ferrous heme rather than its ferric heme. Furthermore, we observed that the heme affinities of these compounds strongly correlate with their IDO1 inhibitory activities.

Soluble guanylate cyclase as an alternative target for bronchodilator therapy in asthma.

Asthma is defined by airway inflammation and hyperresponsiveness, and contributes to morbidity and mortality worldwide. Although bronchodilation is a cornerstone of treatment, current bronchodilators become ineffective with worsening asthma severity. We investigated an alternative pathway that involves activating the airway smooth muscle enzyme, soluble guanylate cyclase (sGC). Activating sGC by its natural stimulant nitric oxide (NO), or by pharmacologic sGC agonists BAY 41-2272 and BAY 60-2770, triggered bronchodilation in normal human lung slices and in mouse airways. Both BAY 41-2272 and BAY 60-2770 reversed airway hyperresponsiveness in mice with allergic asthma and restored normal lung function. The sGC from mouse asthmatic lungs displayed three hallmarks of oxidative damage that render it NO-insensitive, and identical changes to sGC occurred in human lung slices or in human airway smooth muscle cells when given chronic NO exposure to mimic the high NO in asthmatic lung. Our findings show how allergic inflammation in asthma may impede NO-based bronchodilation, and reveal that pharmacologic sGC agonists can achieve bronchodilation despite this loss.

NADH reduction of nitroaromatics as a probe for residual ferric form high-spin in a cytochrome P450.

The existence of a substrate-sensitive equilibrium between high spin (S=5/2) and low spin (S=1/2) ferric iron is a well-established phenomenon in the cytochrome P450 (CYP) superfamily, although its origins are still a subject of discussion. A series of mutations that strongly perturb the spin state equilibrium in the camphor hydroxylase CYP101A1 were recently described (Colthart et al., Sci. Rep. 6, 22035 (2016)). Wild type CYP101A1 as well as some CYP101A1 mutants are herein shown to be capable of catalyzing the reduction of nitroacetophenones by NADH to the corresponding anilino compounds (nitroreductase or NRase activity). The distinguishing characteristic between those mutants that catalyze the reduction and those that cannot appears to be the extent to which residual high spin form exists in the absence of the native substrate d-camphor, with those showing the largest spin state shifts upon camphor binding also exhibiting NRase activity. Optical and EPR spectroscopy was used to further examine these phenomena. These results suggest that reduction of nitroaromatics may provide a useful probe of residual high spin states in the CYP superfamily. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.