Importance of Hydrogen Bonding in Fine Tuning the [2Fe-2S] Cluster Redox Potential of HydC from <i>Thermotoga maritima</i>

Iron–sulfur clusters form one of the largest and most diverse classes of enzyme cofactors in nature. They may serve as structural factors, form electron transfer chains between active sites and external redox partners, or form components of enzyme active sites. Their specific role is a consequence of the cluster type and the surrounding protein environment. The relative effects of these factors are not completely understood, and it is not yet possible to predict the properties of iron–sulfur clusters based on amino acid sequences or rationally tune their properties to generate proteins with new desirable functions. Here, we generate mutations in a [2Fe-2S] cluster protein, the <i>Tm</i>HydC subunit of the trimeric [FeFe]-hydrogenase from <i>Thermotoga maritima</i>, to study the factors that affect its redox potential. Saturation mutagenesis of Val131 was used to tune the redox potential over a 135 mV range and revealed that cluster redox potential and electronic properties correlate with amino acid hydrophobicity and the ability to form hydrogen bonds to the cluster. Proline scanning mutagenesis between pairs of ligating cysteines was used to remove backbone amide hydrogen bonds to the cluster and decrease the redox potential by up to 132 mV, without large structural changes in most cases. However, substitution of Gly83 with proline caused a change of HydC to a [4Fe-4S] cluster protein with a redox potential of −526 mV. Together, these results confirm the importance of hydrogen bonding in tuning cluster redox potentials and demonstrate the versatility of iron–sulfur cluster protein folds at binding different types of clusters

Heterogeneous Kinetics of the Carbon Monoxide Association and Dissociation Reaction to Nitrophorin 4 and 7 Coincide with Structural Heterogeneity of the Gate-Loop

NO is an important signaling molecule in human tissue. However, the mechanisms by which this molecule is controlled and directed are currently little understood. Nitrophorins (NPs) comprise a group of ferriheme proteins originating from blood-sucking insects that are tailored to protect and deliver NO via coordination to and release from the heme iron. Therefore, the kinetics of the association and dissociation reactions were studied in this work using the ferroheme–CO complexes of NP4, NP4­(D30N), and NP7 as isoelectronic models for the ferriheme–NO complexes. The kinetic measurements performed by nanosecond laser-flash-photolysis and stopped-flow are accompanied by resonance Raman and FT-IR spectroscopy to characterize the carbonyl species. Careful analysis of the CO rebinding kinetics reveals that in NP4 and, to a larger extent, NP7 internal gas binding cavities are located, which temporarily trap photodissociated ligands. Moreover, changes in the free energy barriers throughout the rebinding and release pathway upon increase of the pH are surprisingly small in case of NP4. Also in case of NP4, a heterogeneous kinetic trace is obtained at pH 7.5, which corresponds to the presence of two carbonyl species in the heme cavity that are seen in vibrational spectroscopy and that are due to the change of the distal heme pocket polarity. Quantification of the two species from FT-IR spectra allowed the fitting of the kinetic traces as two processes, corresponding to the previously reported open and closed conformation of the A-B and G-H loops. With the use of the A-B loop mutant NP4­(D30N), it was confirmed that the kinetic heterogeneity is controlled by pH through the disruption of the H-bond between the Asp30 side chain and the Leu130 backbone carbonyl. Overall, this first study on the slow phase of the dynamics of diatomic gas molecule interaction with NPs comprises an important experimental contribution for the understanding of the dynamics involved in the binding/release processes of NO/CO in NPs

Expression, Purification, and Solid-State NMR Characterization of the Membrane Binding Heme Protein Nitrophorin 7 in Two Electronic Spin States

The nitrophorins (NPs) comprise a group of NO transporting ferriheme <i>b</i> proteins found in the saliva of the blood sucking insect Rhodnius prolixus. In contrast to other nitrophorins (NP1–4), the recently identified membrane binding isoform NP7 tends to form oligomers and precipitates at higher concentrations in solution. Hence, solid-state NMR (ssNMR) was employed as an alternative method to gain structural insights on the precipitated protein. We report the expression and purification of ¹³C,¹⁵N isotopically labeled protein together with the first ssNMR characterization of NP7. Because the size of NP7 (21 kDa) still provides a challenge for ssNMR, the samples were reverse labeled with Lys and Val to reduce the number of crosspeaks in two-dimensional spectra. The two electronic spin states with <i>S</i> = 1/2 and <i>S</i> = 0 at the ferriheme iron were generated by the complexation with imidazole and NO, respectively. ssNMR spectra of both forms are well resolved, which allows for sequential resonance assignments of 22 residues. Importantly, the ssNMR spectra demonstrate that aggregation does not affect the protein fold. Comparison of the spectra of the two electronic spin states allows the determination of paramagnetically shifted cross peaks due to pseudocontact shifts, which assists the assignment of residues close to the heme center

Unique Spectroscopic Properties of the H‑Cluster in a Putative Sensory [FeFe] Hydrogenase

Sensory type [FeFe] hydrogenases are predicted to play a role in transcriptional regulation by detecting the H₂ level of the cellular environment. These hydrogenases contain the hydrogenase domain with distinct modifications in the active site pocket, followed by a Per-Arnt-Sim (PAS) domain. As yet, neither the physiological function nor the biochemical or spectroscopic properties of these enzymes have been explored. Here, we present the characterization of an artificially maturated, putative sensory [FeFe] hydrogenase from <i>Thermotoga maritima</i> (HydS). This enzyme shows lower hydrogen conversion activity than prototypical [FeFe] hydrogenases and a reduced inhibition by CO. Using FTIR spectroelectrochemistry and EPR spectroscopy, three redox states of the active site were identified. The spectroscopic signatures of the most oxidized state closely resemble those of the H_ox state from the prototypical [FeFe] hydrogenases, while the FTIR spectra of both singly and doubly reduced states show large differences. The FTIR bands of both the reduced states are strongly red-shifted relative to the H_ox state, indicating reduction at the diiron site, but with retention of the bridging CO ligand. The unique functional and spectroscopic features of HydS are discussed with regard to the possible role of altered amino acid residues influencing the electronic properties of the H-cluster

Sonographic visualization of nipple blood flow can help differentiate Paget disease from benign eczematous nipple lesions

<div><p>Purpose</p><p>Paget disease of the breast is a rare cancer that originates from the nipple–areolar complex. It is often overlooked and misdiagnosed as benign chronic eczema of the nipple. We aimed to retrospectively verify whether blood flow analysis using Doppler sonography was useful for detecting the presence of Paget disease.</p><p>Methods</p><p>In this retrospective study, 12 patients with pathologically proven unilateral nipple eczematous lesions (seven with Paget disease and five with simple dermatitis) were included. Nipple blood flow signal was observed using Doppler sonography, and the detected blood flow signals were quantified using digitally recorded images. Quantified blood flow ratio and pathologically examined capillary density were evaluated between affected and unaffected nipples. Findings of mammography, grayscale sonography, and contrast-enhanced magnetic resonance imaging (CE-MRI) were reviewed.</p><p>Results</p><p>In patients with Paget disease, Doppler effects in the affected nipple were more clearly visible than those in the unaffected nipple. These effects were sufficiently visible to identify Paget disease. No obvious effects were observed in the affected and unaffected nipples of simple dermatitis. The quantified blood flow ratio and pathologically examined capillary density were significantly higher for the Paget lesion than those for the non-Paget lesion. The sensitivity of CE-MRI and Doppler sonography was markedly correlated, revealing blood flow changes in the nipple lesions of Paget disease.</p><p>Conclusion</p><p>Doppler sonography visualized the proliferation of blood vessels in Paget lesions. The visualization of increased nipple blood flow using Doppler sonography is a simple and low-cost method that provides useful data for identifying Paget disease during routine medical care.</p></div

Sonographic visualization of nipple blood flow can help differentiate Paget disease from benign eczematous nipple lesions - Fig 3

<p>(a) Visualized hypervascularity of the affected nipple with Paget disease (Case 1). (b) Unchanging blood flow signals of the affected nipple with simple dermatitis (Case 8).</p

Comparison of nipple blood flow ratios between the affected and unaffected nipples with each condition.

<p>Comparison of nipple blood flow ratios between the affected and unaffected nipples with each condition.</p

Histopathological examinations of capillary proliferation of the nipple–areolar region by hematoxylin and eosin staining.

<p>(a) Normal skin control with Paget disease (×40). (b) Normal skin control with Paget disease (×100). (c) Paget lesion at ×40). (d) Paget lesion at ×100). (e) Dermatitis at ×40). (f) Dermatitis at ×100). Bar, 50 μm (b, d, and f).</p

An 81-year-old woman with Paget disease (Case 7).

<p>(a) Scaling and erosion of the affected right nipple. (b) Blood flow signals inside the affected nipple as observed using Doppler sonography. (c) Nipple enhancement by CE-MRI. (d) Histopathological examinations with hematoxylin and eosin staining reveal Paget cells within the epidermis and capillary proliferation within the dermis (×100).</p

The findings of mammography, grayscale sonography, and CE-MRI for seven patients with Paget disease.

<p>The findings of mammography, grayscale sonography, and CE-MRI for seven patients with Paget disease.</p