Thomas A. Link

Isolation and amino acid sequence of the smallest subunit of beef heart bc1 complex

The 11 subunits of beef heart bc 1 complex can be separated either by a new SDS‐PAGE system or by a series of Chromatographic steps involving dissociation of the complex by salt treatment. The amino acid sequence of the smallest subunit was determined by complete solid‐phase Edman degradation and was confirmed by sequencing the N‐terminal part and the C‐terminal tryptic fragment by liquid‐phase Edman degradation. The protein consists of 56 amino acid residues; the M r was calculated to be 6363. The protein (‘ISP binding factor’) might be entangled in the reassembly of the iron‐sulfur protein with the bc 1 subcomplex.

Expression, purification, and characterization of subunit E, an essential subunit of the vacuolar ATPase.

A recombinant form of subunit E (Vma4p) from yeast vacuolar ATPases (V-ATPases) has been overexpressed in Escherichia coli, purified to homogeneity, and explored by mass spectrometry. Analysis of the secondary structure of Vma4p by circular dichroism spectroscopy indicated 32% alpha-helix and 23% beta-sheet content. Vma4p formed a hybrid-complex with the nucleotide-binding subunits alpha and beta of the closely related F(1) ATPase of the thermophilic bacterium PS3 (TF(1)). The alpha(3)beta(3)E-hybrid-complex had 56% of the ATPase activity of the native TF(1). By comparison, an alpha(3)beta(3)-formation without Vma4p showed about 24% of total TF(1) ATPase activity. This is the first demonstration of a hydrolytically active hybrid-complex consisting of F(1) and V(1) subunits. The arrangement of subunit E in V(1) has been probed using the recombinant Vma4p, the alpha(3)beta(3)E-hybrid-complex together with V(1) and an A(3)B(3)HEG-subcomplex of the V(1) ATPase from Manduca sexta, respectively, indicating that subunit E is shielded in V(1).

Molecular cloning and structural analysis of the phosphate translocator from pea chloroplasts and its comparison to the spinach phosphate translocator

Using an 5′-AvaII fragment of the spinach (Spinacia oleracea L.) phosphate translocator cDNA as a probe for a hybridization screening of a pea (Pisum sativum L.) cDNA library we have cloned and sequenced a cDNA clone coding for the phosphate translocator precursor protein from pea chloroplasts. The full-length cDNA clone comprises 42 base pairs (bp) at the 5′-non-coding region, a 1206-bp coding region corresponding to a polypeptide of 402 amino-acid residues (relative molecular mass 43 671) and 244 bp at the non-coding 3′-region. Determination of the N-terminal sequence of the phosphate translocator from both pea and spinach chloroplasts revealed that the transit peptides consist of 72 and 80 amino-acid residues, respectively. These transit peptides are different from those of other chloroplastic transit peptides in that they both contain an amphiphilic α-helix which is located either in close proximity to the processing site in pea or at the N-terminus in spinach. The mature proteins from pea and spinach both contain about 87% identical amino-acid residues and about seven putative membrane-spanning α-helices. Some of these α-helices have an amphiphilic character and might serve to form a hydrophilic translocation channel through the membrane. The in-vitro synthesized pea precursor protein is directed to the chloroplast and inserted into the chloroplast envelope membrane.

Two pK values of the oxidised Rieske [2Fe-2S] cluster observed by CD spectroscopy

Abstract The pH dependence of the CD spectrum of the oxidised ‘Rieske’ [2Fe-2S] cluster was measured over pH = 6.1 to 10.6. The spectral changes could be fitted with two pK values of 7.7 and 9.1. No pH dependent spectral changes were observed on the reduced protein. The far UV CD spectrum of the protein was the same at pH = 6.0 and pH = 10.7; therefore, secondary structure changes are not responsible for the changes in the CD spectrum. It is concluded that the pK values belong to groups directly associated with the [2Fe-2S] cluster, most likely to its histidine ligands.

THE TWO 4FE-4S CLUSTERS IN CHROMATIUM VINOSUM FERREDOXIN HAVE LARGELY DIFFERENT REDUCTION POTENTIALS : STRUCTURAL ORIGIN AND FUNCTIONAL CONSEQUENCES

The 2[4Fe-4S] ferredoxin from Chromatium vinosum arises as one prominent member of a recently defined family of proteins found in very diverse bacteria. The potentiometric circular dichroism titrations of the protein and of several molecular variants generated by site-directed mutagenesis have established that the reduction potentials of the two clusters differ widely by almost 200 mV. This large difference has been confirmed by electrochemical methods, and each redox transition has been assigned to one of the clusters. The unusually low potential center is surprisingly the one that displays a conventional CX 1 X 2CX 3 X 4C (X n , variable amino acid) binding motif and a structural environment similar to that of clusters having less negative potentials. A comparison with other ferredoxins has highlighted factors contributing to the reduction potential of [4Fe-4S] clusters in proteins. (i) The loop between the coordinating cysteines 40 and 49 and the C terminus α-helix of C. vinosum ferredoxin cause a negative, but relatively moderate, shift of ∼60 mV for the nearby cluster. (ii) Very negative potentials, below −600 mV, correlate with the presence of a bulky side chain in positionX 4 of the coordinating triad of cysteines. These findings set the framework in which previous observations on ferredoxins can be better understood. They also shed light onto the possible occurrence and properties of very low potential [4Fe-4S] clusters in less well characterized proteins.

BIOCHEMICAL AND SPECTROSCOPIC PROPERTIES OF THE FOUR-SUBUNIT QUINOL OXIDASE (CYTOCHROME BA3) FROM PARACOCCUS DENITRIFICANS

The ba3 quinol oxidase from Paracoccus denitrificans has been purified by a new protocol leading to significantly higher yields than previously reported (Richter et al. (1994) J. Biol. Chem. 269, 23079-23086). In an SDS PAG an additional protein band compared with the previous preparation appears, which can be identified as the major form of subunit II. All protein bands can be assigned to genes of the qox operon by N-terminal sequencing, indicating that the oxidase consists of four subunits. In addition to one heme A, one heme B, and one copper atom, the preparation contains two ubiquinone molecules per enzyme. The oxidase is further characterized by electron paramagnetic resonance (EPR), circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopy.

Electrochemical study of the redox properties of [2Fe-2S] ferredoxins Evidence for superreduction of the Rieske [2Fe-2S] cluster

Direct, unmediated electrochemistry has been used to compare the redox properties of [2Fe‐2S] clusters in spinach ferredoxin, Spirulina platensis ferredoxin and the water soluble fragment of the Rieske protein. The use of electrochemistry enabled, for the first time, the observation of the second reduction step, [Fe(III),Fe(II)] to [Fe(II),Fe(II)], in a biological [2Fe‐2S] system. A water‐soluble fragment of the Rieske protein from bovine heart bc 1 complex exhibits two subsequent quasi‐reversible responses in cyclic voltammetry on activated glassy carbon. In contrast the ferredoxins from spinach and Spirulina platensis only show one single reduction potential. These results support a seniority scheme for biological iron‐sulfur clusters relating cluster size to electron transfer versatility. Electrochemical reduction of spinach ferredoxin in the presence of NADP+ and ferredoxin: NADP+ oxidoreductase results in the generation of NADPH. The second order rate constant for the reaction between the ferredoxin and the reductase was estimated from cyclic voltammetry experiments to be >3 · 105 M−1 · s−1.

1 – Purification Strategies for Membrane Proteins

Membrane proteins tend to form aggregates even in the presence of detergents, which reduces the efficacy of all separation techniques. It is therefore essential to select an effective solubilizing, but non-denaturing detergent to ascertain the minimum ionic strength the protein requires to be in a homogeneously dissolved state, the maximum ionic strength that will not cause dissociation of labile proteins from complex structures, and the optimum pH for solubilization and protein stability. In planning a strategy for membrane protein isolation, it is necessary to use methods that preserve the native state of the protein in the first steps of the purification. If enzymatic activity is not required, application of denaturing techniques may be advantageous in the final stages when proteins can be identified by apparent molecular masses on SDS-PAGE. Enriching the protein of interest before adding detergents facilitates purification. If there is a choice, attempts should be made to isolate the protein from sources that contain high levels of it.

The structure of the dihaem cytochrome b of fumarate reductase in Wolinella succinogenes : circular dichroism and sequence analysis studies

The fumarate reductase from Wolinella succinogenes contains two haem groups with markedly different midpoint potentials (-20 mV and -200 mV). The enzyme is made up of three subunits, the lipophilic one of which (cytochrome b) ligates the haems. Circular dichroism (CD) spectroscopy has been applied to the reductase in order to obtain information on the structure of the haems and of their environment. This approach is integrated with amino acid sequence comparison of the cytochrome b with other quinone-reacting membrane haemoproteins for predicting the axial ligands of the haems as well as their location relative to the membrane. The following results have been obtained: (1) the CD spectra in the Soret region show exciton coupling indicating haem-haem interaction, which is particularly evident in the reduced state and disappears upon denaturation of the enzyme; (2) The apoprotein of cytochrome b is predicted to consist of five hydrophobic helices (helices A-D and cd), four of which should span the membrane. Helices A, B, C and cd contain a histidine residue each which possibly forms one of the ligands of the haems. It is proposed that haem b (-20 mV) is ligated by H44 and H93, and haem b (-200 mV) by H143 and H182.

Structural and functional analysis of deficient mutants in subunit I of cytochrome c oxidase from Saccharomyces cerevisiae

Four point mutations in subunit I of cytochrome c oxidase from Saccharomyces cerevisiae that had been selected for respiratory incompetence but still contained spectrally detectable haem aa3 were analysed. The isolated mutant enzymes exhibited minor band shifts in their optical spectra and contained all eleven subunits. However, steady state activities were only a few percent compared to wild type enzyme. Using a comprehensive experimental approach, we first checked the integrity of the enzyme preparations and then identified the specific functional defect. The results are discussed using information from the recently solved structures of cytochrome c oxidase at 2.8 A. Mutation 167N is positioned between haem a and a conserved glutamate residue (E243). It caused a distortion of the EPR signal of haem a and shifted its midpoint potential by 54 mV to the negative. The high-resolution structure suggests that the primary reason for the low activity of the mutant enzyme could be that asparagine in position 67 might form a stable hydrogen bond to E243, which is part of a proposed proton channel. Cytochrome c oxidase isolated from mutant T316K did not meet our criteria for homogeneity and was therefore omitted from further analysis. Mutants G352V and V380M exhibited an impairment of electron transfer from haem a to a3 and ligand binding to the binuclear centre was affected. In mutant V380M also the midpoint potential of CuB was shifted by 65 mV to the positive. The results indicated for these two mutants changes primarily associated with the binuclear centre, possibly associated with an interference in the routes and/or sites of protonation which are required for stable formation of the catalytic intermediates. This interpretation is discussed in the light of the high resolution structure.