Assessment of Measurement of Salivary Urea by ATR-FTIR Spectroscopy to Screen for CKD.

Stages of CKD are currently defined by eGFR and require measurement of serum creatinine concentrations. Previous studies have shown a good correlation between salivary and serum urea levels and the stage of CKD. However, quantitative salivary urea assays in current clinical use require costly and labor-intensive commercial kits, which restricts the advantage of using saliva and limits wider applicability as a quick and easy means of assessing renal function.

Electron Transfer Coupled to Conformational Dynamics in Cell Respiration.

Cellular respiration is a fundamental process required for energy production in many organisms. The terminal electron transfer complex in mitochondrial and many bacterial respiratory chains is cytochrome oxidase (CO). This converts the energy released in the cytochrome /oxygen redox reaction into a transmembrane proton electrochemical gradient that is used subsequently to power ATP synthesis.

Are conventional stone analysis techniques reliable for the identification of 2,8-dihydroxyadenine kidney stones? A case series.

We have recently encountered patients incorrectly diagnosed with adenine phosphoribosyltransferase (APRT) deficiency due to misidentification of kidney stones as 2,8-dihydroxyadenine (DHA) stones. The objective of this study was to examine the accuracy of stone analysis for identification of DHA. Medical records of patients referred to the APRT Deficiency Research Program of the Rare Kidney Stone Consortium in 2010-2018 with a diagnosis of APRT deficiency based on kidney stone analysis were reviewed.

A common coupling mechanism for A-type heme-copper oxidases from bacteria to mitochondria.

Mitochondria metabolize almost all the oxygen that we consume, reducing it to water by cytochrome oxidase (CO). CO maximizes energy capture into the protonmotive force by pumping protons across the mitochondrial inner membrane. Forty years after the H/e stoichiometry was established, a consensus has yet to be reached on the route taken by pumped protons to traverse CO's hydrophobic core and on whether bacterial and mitochondrial COs operate via the same coupling mechanism.

The H channel is not a proton transfer path in yeast cytochrome c oxidase.

Cytochrome c oxidases (CcOs) in the respiratory chains of mitochondria and bacteria are primary consumers of molecular oxygen, converting it to water with the concomitant pumping of protons across the membrane to establish a proton electrochemical gradient. Despite a relatively well understood proton pumping mechanism of bacterial CcOs, the role of the H channel in mitochondrial forms of CcO remains debated. Here, we used site-directed mutagenesis to modify a central residue of the lower span of the H channel, Q413, in the genetically tractable yeast Saccharomyces cerevisiae.

Comparison of redox and ligand binding behaviour of yeast and bovine cytochrome c oxidases using FTIR spectroscopy.

Redox and CO photolysis FTIR spectra of yeast cytochrome c oxidase WT and mutants are compared to those from bovine and P. denitrificans CcOs in order to establish common functional features. All display changes that can be assigned to their E242 (bovine numbering) equivalent and to weakly H-bonded water molecules.

Insights into functions of the H channel of cytochrome oxidase from atomistic molecular dynamics simulations.

Proton pumping A-type cytochrome oxidase (CO) terminates the respiratory chains of mitochondria and many bacteria. Three possible proton transfer pathways (D, K, and H channels) have been identified based on structural, functional, and mutational data. Whereas the D channel provides the route for all pumped protons in bacterial A-type COs, studies of bovine mitochondrial CO have led to suggestions that its H channel instead provides this route.

Mitochondrial cytochrome oxidase: catalysis, coupling and controversies.

Mitochondrial cytochrome oxidase is a member of a diverse superfamily of haem-copper oxidases. Its mechanism of oxygen reduction is reviewed in terms of the cycle of catalytic intermediates and their likely chemical structures. This reaction cycle is coupled to the translocation of protons across the inner mitochondrial membrane in which it is located.

Infrared vibrational spectroscopy: a rapid and novel diagnostic and monitoring tool for cystinuria.

Cystinuria is the commonest inherited cause of nephrolithiasis (~1% in adults; ~6% in children) and is the result of impaired cystine reabsorption in the renal proximal tubule. Cystine is poorly soluble in urine with a solubility of ~1 mM and can readily form microcrystals that lead to cystine stone formation, especially at low urine pH. Diagnosis of cystinuria is made typically by ion-exchange chromatography (IEC) detection and quantitation, which is slow, laboursome and costly.

Effects of the Hydration State on the Mid-Infrared Spectra of Urea and Creatinine in Relation to Urine Analyses.

When analyzing solutes by Fourier transform infrared (FT-IR) spectroscopy in attenuated total reflection (ATR) mode, drying of samples onto the ATR crystal surface can greatly increase solute band intensities and, therefore, aid detection of minor components. However, analysis of such spectra is complicated by the existence of alternative partial hydration states of some substances that can significantly alter their infrared signatures. This is illustrated here with urea, which is a dominant component of urine.

Derek Bendall (1930-2014).

Derek Bendall carried out pioneering work on photosynthetic electron transport, particularly on protein-protein interactions, cytochromes, and cyclic electron transport, as well as on other topics including the biochemistry of tea. He was a keen musician and a gifted gardener, a devoted family man, and a delightful colleague and friend. The bioenergetics community, especially those working on photosynthesis, will miss him sorely.

Alkalinity of neutrophil phagocytic vacuoles is modulated by HVCN1 and has consequences for myeloperoxidase activity.

The NADPH oxidase of neutrophils, essential for innate immunity, passes electrons across the phagocytic membrane to form superoxide in the phagocytic vacuole. Activity of the oxidase requires that charge movements across the vacuolar membrane are balanced. Using the pH indicator SNARF, we measured changes in pH in the phagocytic vacuole and cytosol of neutrophils.

Comparisons of subunit 5A and 5B isoenzymes of yeast cytochrome c oxidase.

Subunit 5 of Saccharomyces cerevisiae cytochrome c oxidase (CcO) is essential for assembly and has two isoforms, 5A and 5B. 5A is expressed under normoxic conditions, whereas 5B is expressed at very low oxygen tensions. As a consequence, COX5A-deleted strains (Δcox5A) have no or only low levels of CcO under normoxic conditions rendering them respiratory deficient.

Reaction of wild-type and Glu243Asp variant yeast cytochrome c oxidase with O2.

We have studied internal electron transfer during the reaction of Saccharomyces cerevisiae mitochondrial cytochrome c oxidase with dioxygen. Similar absorbance changes were observed with this yeast oxidase as with the previously studied Rhodobacter sphaeroides and bovine mitochondrial oxidases, which suggests that the reaction proceeds along the same trajectory. However, notable differences were observed in rates and electron-transfer equilibrium constants of specific reaction steps, for example the ferryl (F) to oxidized (O) reaction was faster with the yeast (0.

IR signatures of the metal centres of bovine cytochrome c oxidase: assignments and redox-linkage.

Assignments of IR bands of reduced minus oxidized IR difference spectra of bovine and related cytochrome c oxidases are reviewed and their linkages to specific metal centres are assessed. To aid this, redox-poised difference spectra in the presence of cyanide or carbon monoxide are presented. These ligands fix the redox states of either haem a3 alone or haem a3 and CuB respectively, while allowing redox cycling of the remaining centres.

Functions of the hydrophilic channels in protonmotive cytochrome c oxidase.

The structures and functions of hydrophilic channels in electron-transferring membrane proteins are discussed. A distinction is made between proton channels that can conduct protons and dielectric channels that are non-conducting but can dielectrically polarize in response to the introduction of charge changes in buried functional centres. Functions of the K, D and H channels found in A1-type cytochrome c oxidases are reviewed in relation to these ideas.

Structural changes in cytochrome c oxidase induced by binding of sodium and calcium ions: an ATR-FTIR study.

Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy was used to investigate the binding of Na(+) and Ca(2+)cations to bovine cytochrome c oxidase in its fully oxidized and partially reduced, cyanide-ligated (a(2+)a3(3+)-CN) (mixed valence) forms. These ions induced distinctly different IR binding spectra, indicating that the induced structural changes are different. Despite this, their binding spectra were mutually exclusive, confirming their known competitive binding behavior.

Control of electron transport routes through redox-regulated redistribution of respiratory complexes.

In cyanobacteria, respiratory electron transport takes place in close proximity to photosynthetic electron transport, because the complexes required for both processes are located within the thylakoid membranes. The balance of electron transport routes is crucial for cell physiology, yet the factors that control the predominance of particular pathways are poorly understood. Here we use a combination of tagging with green fluorescent protein and confocal fluorescence microscopy in live cells of the cyanobacterium Synechococcus elongatus PCC 7942 to investigate the distribution on submicron scales of two key respiratory electron donors, type-I NAD(P)H dehydrogenase (NDH-1) and succinate dehydrogenase (SDH).

Assignment of the CO-sensitive carboxyl group in mitochondrial forms of cytochrome c oxidase using yeast mutants.

Point mutations of E243D and I67N were introduced into subunit I of a 6histidine-tagged (6H-WT) form of yeast Saccharomyces cerevisiae mitochondrial cytochrome c oxidase. The two mutants (6H-E243D(I) and 6H-I67N(I)) were purified and showed ≈50 and 10% of the 6H-WT turnover number. Light-induced CO photolysis FTIR difference spectra of the 6H-WT showed a peak/trough at 1749/1740cm(-1), as seen in bovine CcO, which downshifted by 7cm(-1) in D(2)O.

Construction of histidine-tagged yeast mitochondrial cytochrome c oxidase for facile purification of mutant forms.

Yeast CcO (cytochrome c oxidase) has been developed as a facile system for the production and analysis of mutants of a mitochondrial form of CcO for mechanistic studies. First, a 6H tag (His6 tag) was fused to the C-terminus of a nuclear-encoded subunit of CcO from yeast Saccharomyces cerevisiae. This allowed efficient purification of a WT (wild-type) mitochondrial CcO, 6H-WT (yeast CcO with a 6H tag on the nuclear-encoded Cox13 subunit), with a recovery yield of 45%.

Yeast cytochrome c oxidase: a model system to study mitochondrial forms of the haem-copper oxidase superfamily.

The known subunits of yeast mitochondrial cytochrome c oxidase are reviewed. The structures of all eleven of its subunits are explored by building homology models based on the published structures of the homologous bovine subunits and similarities and differences are highlighted, particularly of the core functional subunit I. Yeast genetic techniques to enable introduction of mutations into the three core mitochondrially-encoded subunits are reviewed.

Water molecule reorganization in cytochrome c oxidase revealed by FTIR spectroscopy.

Although internal electron transfer and oxygen reduction chemistry in cytochrome c oxidase are fairly well understood, the associated groups and pathways that couple these processes to gated proton translocation across the membrane remain unclear. Several possible pathways have been identified from crystallographic structural models; these involve hydrophilic residues in combination with structured waters that might reorganize to form transient proton transfer pathways during the catalytic cycle. To date, however, comparisons of atomic structures of different oxidases in different redox or ligation states have not provided a consistent answer as to which pathways are operative or the details of their dynamic changes during catalysis.

The mitochondrial respiratory chain.

In the present chapter, the structures and mechanisms of the major components of mammalian mitochondrial respiratory chains are reviewed. Particular emphasis is placed on the four protein complexes and their cofactors that catalyse the electron transfer pathway between oxidation of NADH and succinate and the reduction of oxygen to water. Current ideas are reviewed of how these electron transfer reactions are coupled to formation of the proton and charge gradient across the inner mitochondrial membrane that is used to drive ATP synthesis.