Impact of residues remote from the catalytic centre on enzyme catalysis of copper nitrite reductase

Enzyme mechanisms are often probed by structure-informed point mutations and measurement of their effects on enzymatic properties to test mechanistic hypotheses. In many cases, the challenge is to report on complex, often inter-linked elements of catalysis. Evidence for long-range effects on enzyme mechanism resulting from mutations remains sparse, limiting the design/redesign of synthetic catalysts in a predictable way. Here we show that improving the accessibility of the active site pocket of copper nitrite reductase by mutation of a surface-exposed phenylalanine residue (Phe306), located 12 Å away from the catalytic site type-2 Cu (T2Cu), profoundly affects intra-molecular electron transfer, substrate-binding and catalytic activity. Structures and kinetic studies provide an explanation for the lower affinity for the substrate and the alteration of the rate-limiting step in the reaction. Our results demonstrate that distant residues remote from the active site can have marked effects on enzyme catalysis, by driving mechanistic change through relatively minor structural perturbations

Occupational Therapists as Responders in Disaster Management

Purpose: The quantitative study explored the awareness, participation in disaster management stages, training, relationship of practice settings to likelihood of responding, and difference between occupational therapists (OTs) and occupational therapy assistants (OTAs) likelihood of responding in each stage of disaster management. Method: A Healthcare Practitioner Disaster Management Questionnaire was used via REDCap survey system to survey licensed OTs and licensed OTAs actively practicing in the state of Florida. Data were collected through convenience sampling through the Florida Department of Health public portal download. A review of personal and professional demographic descriptive data indicated the 282 OTs and 115 OTAs responded similarly. Results: Goodness of fit tests indicated that the study population was similar to the AOTA workforce population for median age but not practice setting or median years of practice. A chi-square test indicated participants were not aware of opportunities to participate in disaster management stages. A descriptive analysis review found OTs and OTAs lack involvement in disaster management stages. A descriptive analysis identified a limited number of practitioners received training to enable participation in the stages of disaster management. The results from Mann-Whitney U analyses suggested OTAs are more likely than OTs to respond to different types of disasters. Eta squared statistics for OTs and OTAs indicated no relationship between practice setting and likelihood of responding to disasters. Conclusions and Relevance: This study found that OT practitioners have the capability to respond within the different stages of disaster management, however, there is a lack of training and involvement in disaster response. This study contributes to the literature in occupational therapy to increase awareness of the lack of knowledge, training, and involvement of occupational therapists in the disaster management stages. Hopefully, this study provides a wake-up call to educators of the need to provide knowledge of disaster management in the training of new OT practitioners, continuing education providers of the need to offer disaster management training to practicing therapists, and all OT practitioners of the need to be involved in the health and welfare of people and communities they live in as well as around the world

Proton-Coupled Electron Transfer in the Catalytic Cycle of Alcaligenes xylosoxidans Copper-Dependent Nitrite Reductase

We demonstrated recently that two protons are involved in reduction of nitrite to nitric oxide through a proton-coupled electron transfer (ET) reaction catalyzed by the blue Cu-dependent nitrite reductase (Cu NiR) of Alcaligenes xylosoxidans (AxNiR). Here, the functionality of two putative proton channels, one involving Asn90 and the other His254, is studied using single (N90S, H254F) and double (N90S-H254F) mutants. All mutants studied are active, indicating that protons are still able to reach the active site. The H254F mutation has no effect on the catalytic activity, while the N90S mutation results in ∼70% decrease in activity. Laser flash-photolysis experiments show that in H254F and wild-type enzyme electrons enter at the level of the T1Cu and then redistribute between the two Cu sites. Complete ET from T1Cu to T2Cu occurs only when nitrite binds at the T2Cu site. This indicates that substrate binding to T2Cu promotes ET from T1Cu, suggesting that the enzyme operates an ordered mechanism. In fact, in the N90S and N90S-H254F variants, where the T1Cu site redox potential is elevated by ∼60 mV, inter-Cu ET is only observed in the presence of nitrite. From these results it is evident that the Asn90 channel is the main proton channel in AxNiR, though protons can still reach the active site if this channel is disrupted. Crystallographic structures provide a clear structural rationale for these observations, including restoration of the proton delivery via a significant movement of the loop connecting the T1Cu ligands Cys130 and His139 that occurs on binding of nitrite. Notably, a role for this loop in facilitating interaction of cytochrome c551 with Cu NiR has been suggested previously based on a crystal structure of the binary complex. © 2011 American Chemical Society

Gating mechanisms for biological electron transfer: Integrating structure with biophysics reveals the nature of redox control in cytochrome P450 reductase and copper-dependent nitrite reductase

Edited by Miguel Teixeira and Ricardo O. Louro Keywords: Electron transfer Gating Proton coupled electron transfer Conformationally controlled electron transfer a b s t r a c t Biological electron transfer is a fundamentally important reaction. Despite the apparent simplicity of these reactions (in that no bonds are made or broken), their experimental interrogation is often complicated because of adiabatic control exerted through associated chemical and conformational change. We have studied the nature of this control in several enzyme systems, cytochrome P450 reductase, methionine synthase reductase and copper-dependent nitrite reductase. Specifically, we review the evidence for conformational control in cytochrome P450 reductase and methionine synthase reductase and chemical control i.e. proton coupled electron transfer in nitrite reductase. This evidence has accrued through the use and integration of structural, spectroscopic and advanced kinetic methods. This integrated approach is shown to be powerful in dissecting control mechanisms for biological electron transfer and will likely find widespread application in the study of related biological redox systems

Examining the role of the school Professional Experience Coordinator in the NSW Department of Education’s Professional Experience Hub School program : Final report for the NSW Department of Education

This document reports the evidence gathered in a research study that critically examined the role of the school Professional Experience Coordinator in supporting the professional experience component of initial teacher education courses. The focus of the study was the school Professional Experience Coordinators involved in the 2016-2018 and 2019-2021 cohorts of the NSW Department of Education’s Professional Experience Hub School program. This study was funded by the NSW Department of Education and undertaken under contract by the NSW Council of Deans of Education. The purpose of the Hub Schools has been summarised by the Department’s own Centre for Educational Statistics and Evaluation (2018, p7) as: Hub Schools have introduced initiatives that are targeted at both preservice teachers and supervising teachers. For preservice teachers, these most commonly include revised supervising models, increased support structures such as induction and orientation sessions, professional learning, and additional resources. For supervising teachers, initiatives include the provision of professional learning and additional support. Initiatives that benefitted partner schools include the provision of professional learning, funding, resources and increased support. Other initiatives and outcomes include revisions to Initial Teacher Education (ITE) course content and modified content delivery. METHOD The study was qualitative and used a quasi-narrative approach which collected accounts of the phenomenon in question from the perspective of the participants. The participants were the 24 Professional Experience Coordinators at the hub schools, their Principals (n=20) and the coordinator of professional experience at their respective partner university (n=12). The research method was appropriate for the research questions as it sought to understand and reflect the specific experiences of these three groups within the specific context of the Professional Experience Hub School program. An evaluation of the Hub School program more broadly is beyond the scope of this study. RESEARCH QUESTIONS The key questions that were addressed through this evaluation are: 1. What is the role of the Professional Experience Coordinator in enhancing professional experience in teacher education? 2. What strategies do Professional Experience Coordinators, Principals and University Coordinators in partner universities see as supporting them to develop and maintain the ongoing quality of the Professional Experience Hub School program? Executive Summary Examining the Role of the Professional Experience Coordinator in the NSW Department of Education’s Professional Experience Hub School Program