The diheme enzyme MauG catalyzes the posttranslational modification of the precursor protein of methylamine dehydrogenase (preMADH) to complete biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. Catalysis proceeds through a high valent bis-Fe(IV) redox state and requires long-range electron transfer (ET), as the distance between the modified residues of pre-MADH and the nearest heme iron of MauG is 19.4 Å. Trp199 of MauG resides at the MauG-preMADH interface, positioned midway between the residues that are modified and the nearest heme. W199F and W199K mutations did not affect the spectroscopic and redox properties of MauG, or its ability to stabilize the bis-Fe(IV) state. Crystal structures of complexes of W199F/K MauG with pre-MADH showed no significant perturbation of the MauG-preMADH structure or protein interface. However, neither MauG variant was able to synthesize TTQ from preMADH. In contrast, an ET reaction from diferrous MauG to quinone MADH, which does not require the bis-Fe(IV) intermediate, was minimally affected by the W199F/K mutations. W199F/K MauGs were able to oxidize quinol MADH to form TTQ, the putative final two-electron oxidation of the biosynthetic process, but with k cat ∕K m values approximately 10% that of wild-type MauG. The differential effects of the W199F/K mutations on these three different reactions are explained by a critical role for Trp199 in mediating multistep hopping from preMADH to bis-Fe(IV) MauG during the long-range ET that is required for TTQ biosynthesis.cytochrome | electron hopping | peroxidase | protein oxidation | protein radical L ong-range electron transfer (ET) through proteins is required for biological processes including respiration, photosynthesis, and metabolism. Mechanisms by which ET occurs over large distances to specific sites within a protein have been extensively studied (1-4). For interprotein ET, kinetic mechanisms are more complex, as the overall redox reaction requires additional steps such as protein-protein association and reorientation of the protein complex to optimize the system for ET (5, 6). "Long-range catalysis" is a related process in which the redox center that provides the oxidizing or reducing power is physically distinct from the site of chemical reaction of the substrate, so that long-range ET is required for catalysis. Thus far two enzymes have been postulated to employ long-range catalysis. Ribonucleotide reductase (RNR) catalyzes the formation of deoxyribonucleotides from ribonucleotides by long-range ET via multiple tyrosyl residues (7,8). DNA photolyase is a flavoprotein that catalyzes DNA repair of pyrimidine-pyrimidine dimers via multiple tryptophan residues (9). In these enzymes it is believed that the long-range ET proceeds by hopping (10) through residues that can stabilize a radical state, rather than via a single long-range electron tunneling event.

The diheme enzyme MauG catalyzes the posttranslational modification of a precursor protein of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. It catalyzes three sequential two-electron oxidation reactions which proceed through a high valent bis-Fe(IV) redox state. Tyr294, the unusual distal axial ligand of one c-type heme, was mutated to His and the crystal structure of Y294H MauG in complex with preMADH reveals that this heme now has His-His axial ligation. Y294H MauG is able to interact with preMADH and participate in inter-protein electron transfer, but it is unable to catalyze the TTQ biosynthesis reactions that require the bis-Fe(IV) state. This mutation not only affects the redox properties of the six-coordinate heme but also the redox and CO-binding properties of the five-coordinate heme, despite the 21 Å separation of the heme iron centers. This highlights the communication between the hemes which in wild-type MauG behave as a single diheme unit. Spectroscopic data suggest that Y294H MauG can stabilize a high valent redox state equivalent to Fe(V), but it appears to be an Fe(IV)=O/π radical at the five-coordinate heme rather than the bis-Fe(IV) state. This compound I-like intermediate does not catalyze TTQ biosynthesis, demonstrating that the bis-Fe(IV) state, which is stabilized by Tyr294, is specifically required for this reaction. The TTQ biosynthetic reactions catalyzed by wild-type MauG do not occur via direct contact with the Fe(IV)=O heme, but via long range electron transfer through the six-coordinate heme. Thus, a critical feature of the bis-Fe(IV) species may be that it shortens the electron transfer distance from preMADH to a high valent heme iron.

The diheme enzyme MauG catalyzes the posttranslational modification of a precursor protein of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. This six-electron oxidation of preMADH requires long range electron transfer (ET) as the structure of the MauG-preMADH complex reveals that the shortest distance between the modified residues of preMADH and the nearest heme of MauG is ET from diferrous MauG to oxidized TTQ of MADH exhibits a K d of 10.1 μM and rate constant of 0.07 s −1 . These similar K d values are much greater than that for the MauG-preMADH complex, indicating that the extent of TTQ maturity rather than its redox state influences complex formation. The difference in rate constants is consistent with a larger driving force for the faster reaction. Analysis of the structure of the MauG-preMADH complex in the context of ET theory and these results suggests that direct electron tunneling between the residues which form TTQ and the five-coordinate oxygen-binding heme is not possible, and that ET requires electron hopping via the six-coordinate heme.Methylamine dehydrogenase (MADH)1 is a 119 kDa heterotetrameric α 2 β 2 protein (1,2) with each β subunit possessing a tryptophan tryptophylquinone (TTQ) (3) protein-derived cofactor (4). TTQ is formed by post-translational modification of two tryptophan residues of the polypeptide chain. For TTQ biosynthesis, two atoms of oxygen are incorporated into the indole ring of βTrp57 and a covalent bond is formed between the indole rings of βTrp57 and βTrp108. TTQ biosynthesis requires the action of MauG (5) which is a 42.3 kDa enzyme containing two c-type hemes (6). MauG exhibits homology to diheme cytochrome c peroxidase (7,8), but displays significant differences in catalytic and redox behavior (9,10). Inactivation of mauG, a gene in the methylamine utilization (mau) gene cluster (11) leads to accumulation of a biosynthetic precursor protein (preMADH) in which βTrp57 is monohydroxylated at C7 position, and the covalent crosslink between βTrp57 and βTrp108 is absent (12,13 His205 and Tyr294 (15). While the order is not known, the overall process of MauG-dependent TTQ biosynthesis requires three sequential two-electron oxidations to catalyze the second hydroxylation of βTrp57, the crosslink formation between βTrp57 and βTrp108, and the oxidation to the quinone state.As TTQ is a two-electron redox cofactor, it may be present in MADH in three different redox states. The fully oxidized quinone exhibits a broad absorbance centered at 440 nm, the one-electron reduced semiquinone exhibits an absorption maximum at 428 nm, and the fully reduced quinol exhibits an absorption maximum at 330 nm (16 The kinetic mechanism of initial two-electron oxidation of preMADH by bis-Fe(IV) MauG was previously characterized (17). The reaction exhibited a random kinetic mechanism, in which the order of addition of [O] and preMADH did not matter. Such a random mechanism is in contrast to that typically...

The diheme enzyme MauG catalyzes a six-electron oxidation required for posttranslational modification of a precursor of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. Crystallographic studies have implicated Glu113 in the formation of the bis-FeIV state of MauG, in which one heme is FeIV=O and the other is FeIV with His-Tyr axial ligation. An E113Q mutation had no effect on the structure of MauG, but significantly altered its redox properties. E113Q MauG could not be converted to the diferrous state by reduction with dithionite, but was only reduced to a mixed valence FeII/FeIII state, which is never observed in wild-type (WT) MauG. Addition of H2O2 to E113Q MauG generated a high valence state that formed more slowly and was less stable than the bis-FeIV state of WT MauG. E113Q MauG exhibited no detectable TTQ biosynthesis activity in a steady-state assay with preMADH as the substrate. It did catalyze the steady-state oxidation of quinol MADH to the quinone, but 1000-fold less efficiently than WT MauG. Addition of H2O2 to a crystal of the E113Q MauG-preMADH complex resulted in partial synthesis of TTQ. Extended exposure of these crystals to H2O2 resulted in hydroxylation of Pro107 in the distal pocket of the high-spin heme. It is concluded that the loss of the carboxylic group of Glu113 disrupts the redox cooperativity between hemes that allows rapid formation of the diferrous state, and alters the distribution of high-valence species that participate in charge-resonance stabilization of the bis-FeIV redox state.

The purpose of this study is to examine factors that influence the diagnostic ability of dental students with regards to oral cancer and oral potentially malignant disorders. Dental students at different levels of study were directly interviewed to examine their oral cancer knowledge and diagnostic ability using a validated and pre-tested survey instrument containing validated clinical images of oral cancer and oral potentially malignant disorders. An oral cancer knowledge scale (0 to 31) was generated from correct responses on oral cancer general knowledge, and a diagnostic ability scale (0 to 100) was generated from correct selections of suspicious oral lesions. Knowledge scores ranged from 0 to 27 (mean 10.1 ± 6.0); mean knowledge scores increased with year of study; 5th year students had the highest mean knowledge score (19.1 ± 4.0), while 1st year students had the lowest (5.6 ± 3.5). Diagnostic ability scores increased with year of study and ranged from 0 to 88.5 % (mean 41.8 % ± 15.6). The ability to recognize suspicious oral lesions was significantly correlated with knowledge about oral cancer and oral potentially malignant disorders (r = 0.28; P < 0.001). There is a need to improve oral cancer education curricula; increasing students' contact with patients who have oral lesions including oral cancer will help to improve their future diagnostic ability and early detection practices.

Prostate cancer (PCA) is one of the most common cancer types in men, with cancer progression being linked to hypoxia and the induction of hypoxia-inducible factor (HIF).We investigated the expression of pyruvate kinase M2 (PKM2), its regulation by HIF isoforms 1α and 2α, and its role in HIF stabilization. We additionally examined cell survival in the prostate cancer cell lines PC3 and LNCaP under severe hypoxic (0.1% O2) and normoxic (20% O2) conditions. qRT-PCR showed higher up-regulation of PKM2 mRNA expression in LNCaP cells than in PC3 cells, while western blotting showed that PKM2 protein levels were up-regulated only in LNCaP cells. Inhibition of HIF-1α and HIF-2α by small interfering RNA (si-RNA) demonstrated HIF-1α dependent up-regulation of PKM2 at the mRNA and protein levels in LNCaP cells. PKM2 inhibition by si-RNA significantly decreased hypoxia-response element (HRE) activation in a gene reporter assay and down-regulated HIF-1α target vascular endothelial growth factor (VEGF) mRNA expression in PC3 cells, whereas HIF-1α protein levels were not significantly reduced. Additionally, PKM2 inhibition significantly reduced clonogenic survival in both cell lines in a colony formation assay. Prolyl hydroxylase 3 (PHD3) mRNA expression was up-regulated in both cell lines. It has been shown that PKM2 expression is regulated by HIF-1α and that PKM2 favors HIF-1α transactivation under mild (1% O2) but not severe (0.1% O2) hypoxic conditions, and some of our findings are consistent with these previous results. However, this mechanism was not fully observed in our studied cell lines, as PKM2 regulation and HIF-1α stabilization at the transactivation level occurred under severe hypoxic conditions. This discrepancy suggests that tumor tissue origin and cell type influence this model. Our findings expand the current knowledge of the mechanisms of PCA regulation, and would be important in developing novel therapeutic strategies.

MauG catalyzes posttranslational modifications of methylamine dehydrogenase to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. MauG possesses a five-coordinate high-spin and a six-coordinate low-spin ferric heme, the latter with His-Tyr ligation. Replacement of this tyrosine with lysine generates a MauG variant with only high-spin ferric heme and altered spectroscopic and redox properties. Y294K MauG cannot stabilize the bis-Fe(IV) redox state required for TTQ biosynthesis but instead forms a compound I-like species on reaction with peroxide. The results clarify the role of Tyr ligation of the five-coordinate heme in determining the physical and redox properties and reactivity of MauG.

Background Research ethics is required for high-quality research that positively influences society. There is limited understanding of research ethics in Middle Eastern countries including Jordan. Here, we aim to investigate the level of understanding of research ethics principles among health sciences faculty members in Jordan. Methods This is a cross sectional study where faculty members from the University of Jordan were surveyed for their knowledge and, attitude of research ethics principles. The study was conducted in the period between July 2016 to July 2017 using a customized-design questionnaire involving demographic data and participants’ contributions toward research, and assessment of participants’ knowledge, belief and attitude towards research ethics. Different question-formats have been used including multiple-choice, yes or no, and a four point Likert-type questions. Obtained responses were tabulated according to gender, academic-rank, and knowledge about research ethics principles. Results The study had a response rate of 51%. Among the 137 participants of this study, most (96%) were involved in human and animal research, yet, only 2/3 had prior training in research ethics. Moreover, 91% believed that investigators should have training in research ethics and 87% believed that there should be a mandatory postgraduate course on that. The average correct scores for correct understanding of researchers towards research ethics was 62%. Yet, there were some misconceptions about the major ethical principles as only 43% identified them correctly. Additionally, the role of research ethics committees was not well understood by most of the respondents. Conclusions Although there is acceptable knowledge about research ethics, discrepancies in understanding in research ethics principles seems to exist. There is a large support for further training in responsible conduct of research by faculty in health sciences in Jordan. Thus, such training should be required by universities to address this knowledge gap in order to improve research quality and its impact on society.