Histidine is the axial ligand to cytochrome alpha 3 in cytochrome c oxidase

The nitric oxide-bound complexes of reduced yeast cytochrome c oxidase incorporated with [1,3-15N2]histidine have been investigated by EPR spectroscopy. The results of this study have allowed the unambiguous identification of histidine as the endogenous axial ligand to cytochrome alpha 3

Structures and proton-pumping strategies of mitochondrial respiratory enzymes

Enzymes of the mitochondrial respiratory chain serve as proton pumps, using the energy made available from electron transfer reactions to transport protons across the inner mitochondrial membrane and create an electrochemical gradient used for the production of ATP. The ATP synthase enzyme is reversible and can also serve as a proton pump by coupling ATP hydrolysis to proton translocation. Each of the respiratory enzymes uses a different strategy for performing proton pumping. In this work, the strategies are described and the structural bases for the action of these proteins are discussed in light of recent crystal structures of several respiratory enzymes. The mechanisms and efficiency of proton translocation are also analyzed in terms of the thermodynamics of the substrate transformations catalyzed by these enzymes

Proton translocation in proteins

The active transport of protons across the low dielectric barrier imposed by biological membranes is accomplished by a plethora of proteins that span the ca. 40 Å of the phospholipid bilayer. The free energy derived from the proton electrochemical potential established by the translocation of these protons can subsequently be used to drive vital chemical reactions of the cell, such as ATP synthesis and cell locomotion. Membrane-bound proton translocating proteins have now been found for a variety of organisms and tissues (1). The driving force for proton pumping in these proteins is supplied by numerous mechanisms, including light absorption (e.g. bacteriorhodopsin) (2a,b), ligand binding (e.g. ATPase) (3), and electrochemistry (e.g. electron transfer through cytochrome c oxidase) (4). Thus nature has devised a variety of methods for supplying the energy required for proton pumping by these proteins. Such diversity notwithstanding, the proteins most likely share some common elements of structure and mechanism that allow them to function as proton pumps. A number of theoretical mechanisms have been put forth for both general proton translocation (5-7) and for energy coupling in specific proton pumps. However, despite almost three decades of intensive research, the details of the mechanism(s) and structural requirements for proton pumping remain largely unresolved. To some extent this is the result of the paucity of structural information available for integral membrane proteins. This situation may soon improve as a result of advances in protein methodologies that have allowed several integral membrane proteins to be successfully crystalized (8), and the increased use of genetic engineering to obtain recombinant proton translocating proteins that will offer an opportunity to assess the importance of specific amino acids for the proton translocation process (9)

Characterization of the transverse relaxation rates in lipid bilayers

The 2H NMR transverse relaxation rates of a deuterated phospholipid bilayer reflect slow motions in the bilayer membrane. A study of dimyristoyl lecithin specifically deuterated at several positions of the hydrocarbon chains indicates that these motions are cooperative and are confined to the hydrocarbon chains of the lipid bilayer. However, lipid head group interactions do play an important role in modulating the properties of the cooperative fluctuations of the hydrocarbon chains (director fluctuations), as evidenced by the effects of various lipid additives on the 2H NMR transverse relaxation rates of the dimyristoyl lecithin bilayer

NMR studies of membrane structure and dynamics

Over the past decade, there has been considerable interest in the motional state of the phospholipid bilayer membrane. The motivation underlying these efforts has been the contention that the phospholipid bilayer is the basic matrix in which membrane proteins are embedded to form the biological membrane, and that the permeability and mechanical properties of the membrane, as well as the enzymatic activity of membrane proteins, are dependent upon the fluidity of the bilayer, especially the motional state of the hydrocarbon chains

The minimal structure containing the band 3 anion transport site. A 35Cl NMR study

35Cl NMR, which enables observation of chloride binding to the anion transport site on band 3, is used in the present study to determine the minimal structure containing the intact transport site. Removal of cytoskeletal and other nonintegral membrane proteins, or removal of the 40-kDa cytoskeletal domain of band 3, each leave the transport site intact. Similarly, cleavage of the 52-kDa transport domain into 17- and 35-kDa fragments by chymotrypsin leaves the transport site intact. Extensive proteolysis by papain reduces the integral red cell membrane proteins to their transmembrane segments. Papain treatment removes approximately 60% of the extramembrane portion of the transport domain and produces small fragments primarily in the range 3-7 kDa, with 5 kDa being most predominant. Papain treatment damages, but does not destroy, chloride binding to the transport site; thus, the minimal structure containing the transport site is composed solely of transmembrane segments. In short, the results are completely consistent with a picture in which the transport site is buried in the membrane where it is protected from proteolysis; the transmembrane segments that surround the transport site are held together by strong attractive forces within the bilayer; and the transport site is accessed by solution chloride via an anion channel leading from the transport site to the solution

The identification of histidine ligands to cytochrome a in cytochrome c oxidase

A histidine auxotroph of Saccharomyces cerevisiae has been used to metabolically incorporate [1,3-15N2] histidine into yeast cytochrome c oxidase. Electron nuclear double resonance (ENDOR) spectroscopy of cytochrome a in the [15N]histidine-substituted enzyme reveals an ENDOR signal which can be assigned to hyperfine coupling of a histidine 15N with the low-spin heme, thereby unambiguously identifying histidine as an axial ligand to this cytochrome. Comparison of this result with similar ENDOR data obtained on two 15N-substituted bisimidazole model compounds, metmyoglobin-[15N]imidazole and bis[15N]imidazole tetraphenyl porphyrin, provides strong evidence for bisimidazole coordination in cytochrome a

Chloride binding to the anion transport binding sites of band 3. A 35Cl NMR study

Band 3 is an integral membrane protein that exchanges anions across the red cell membrane. Due to the abundance and the high turnover rate of the band 3 transport unit, the band 3 system is the most heavily used ion-transport system in a typical vertebrate organism. Here we show that 35Cl NMR enables direct and specific observation of substrate Cl- binding to band 3 transport sites, which are identified by a variety of criteria: (a) the sites are inhibited by 4,4'- dinitrostilbene -2,2'- disulfonate, which is known to inhibit competitively Cl- binding to band 3 transport sites; (b) the sites have affinities for 4,4'- dinitrostilbene -2,2'-disulfonate and Cl- that are quantitatively similar to the known affinities of band 3 transport sites for these anions; and (c) the sites have relative affinities for Cl-, HCO-3, F-, and I- that are quantitatively similar to the known relative affinities of band 3 transport sites for these anions. The 35Cl NMR assay also reveals a class of low affinity Cl- binding sites (KD much greater than 0.5 M) that are not affected by 4,4'- dinitrostilbene -2,2'- disulfonate. These low affinity sites may be responsible for the inhibition of band 3 catalyzed anion exchange that has been previously observed at high [Cl-]. In the following paper the 35Cl NMR assay is used to resolve the band 3 transport sites on opposite sides of the membrane, thereby enabling direct observation of the transmembrane recruitment of transport sites

Spectroscopic characterization of the oxo-transfer reaction from a bis(µ-oxo)dicopper(III) complex to triphenylphosphine

The oxygen-atom transfer reaction from the bis(µ-oxo)dicopper(III) complex [CuIII2(µ-O)2(L)2]2+1, where L =N,N,N,N -tetraethylethylenediamine, to PPh3 has been studied by UV-vis, EPR, 1H NMR and Cu K-edge X-ray absorption spectroscopy in parallel at low temperatures (193 K) and above. Under aerobic conditions (excess dioxygen), 1 reacted with PPh3, giving OPPh3 and a diamagnetic species that has been assigned to an oxo-bridged dicopper(II) complex on the basis of EPR and Cu K-edge X-ray absorption spectroscopic data. Isotope-labeling experiments (18O2) established that the oxygen atom incorporated into the triphenylphosphine oxide came from both complex 1 and exogenous dioxygen. Detailed kinetic studies revealed that the process is a third-order reaction; the rate law is first order in both complex 1 and triphenylphosphine, as well as in dioxygen. At temperatures above 233 K, reaction of 1 with PPh3 was accompanied by ligand degradation, leading to oxidative N-dealkylation of one of the ethyl groups. By contrast, when the reaction was performed in the absence of excess dioxygen, negligible substrate (PPh3) oxidation was observed. Instead, highly symmetrical copper complexes with a characteristic isotropic EPR signal at g= 2.11 were formed. These results are discussed in terms of parallel reaction channels that are activated under various conditions of temperature and dioxygen