Crystallographic studies of the Escherichia coli quinol-fumarate reductase with inhibitors bound to the quinol-binding site

The quinol-fumarate reductase (QFR) respiratory complex of Escherichia coli is a four-subunit integral-membrane complex that catalyzes the final step of anaerobic respiration when fumarate is the terminal electron acceptor. The membrane-soluble redox-active molecule menaquinol (MQH(2)) transfers electrons to QFR by binding directly to the membrane-spanning region. The crystal structure of QFR contains two quinone species, presumably MQH(2), bound to the transmembrane-spanning region. The binding sites for the two quinone molecules are termed Q(P) and Q(D), indicating their positions proximal Q(P)) or distal (Q(D)) to the site of fumarate reduction in the hydrophilic flavoprotein and iron-sulfur protein subunits. It has not been established whether both of these sites are mechanistically significant. Co-crystallization studies of the E. coli QFR with the known quinol-binding site inhibitors 2-heptyl-4-hydroxyquinoline-N-oxide and 2-[1-(p-chlorophenyl)ethyl] 4,6-dinitrophenol establish that both inhibitors block the binding of MQH(2) at the Q(P) site. In the structures with the inhibitor bound at Q(P), no density is observed at Q(D), which suggests that the occupancy of this site can vary and argues against a structurally obligatory role for quinol binding to Q(D). A comparison of the Q(P) site of the E. coli enzyme with quinone-binding sites in other respiratory enzymes shows that an acidic residue is structurally conserved. This acidic residue, Glu-C29, in the E. coli enzyme may act as a proton shuttle from the quinol during enzyme turnover

Helix Dipole Movement and Conformational Variability Contribute to Allosteric GDP Release in Gα i Subunits † , ‡

Heterotrimeric G proteins (Gαβγ) transmit signals from activated G protein coupled receptors (GPCRs) to downstream effectors through a guanine nucleotide signaling cycle. Numerous studies indicate that the carboxy-terminal α5 helix of Gα subunits participate in Gα-receptor binding, and previous EPR studies suggest this receptor-mediated interaction induces a rotation and translation of the α5 helix of the Gα subunit [Oldham et al., Nat. Struct. Mol. Biol., 13: 772-7 (2006)]. Based on this result, an engineered disulfide bond was designed to constrain the α5 helix of Gαi1 into its EPR-measured receptor-associated conformation through the introduction of cysteines at positions 56 in the α1 helix and 333 in the α5 helix (I56C/Q333C Gαi1). A functional mimetic of the EPR-measured α5 helix dipole movement upon receptor association was additionally created by introduction of a positive charge at the amino-terminus of this helix, D328R Gαi1. Both proteins exhibit dramatically elevated basal nucleotide exchange. The 2.9 Å resolution crystal structure of the I56C/Q333C Gαi1 in complex with GDP-AlF4− reveals the shift of the α5 helix toward the guanine nucleotide-binding site that is anticipated by EPR measurements. The structure of the I56C/Q333C Gαi1 subunit further revealed altered positions for the switch regions and throughout the Gαi1 subunit, accompanied by significantly elevated crystallographic temperature factors. Combined with previous evidence in the literature, the structural analysis supports the critical role of electrostatics of the α5 helix dipole and overall conformational variability during nucleotide release

High-Resolution Structures of the Oxidized and Reduced States of Cytochrome c554 from Nitrosomonas europaea

Cytochrome c554 (cyt c554) is a tetra-heme cytochrome involved in the oxidation of NH3 by Nitrosomonas europaea. The X-ray crystal structures of both the oxidized and dithionite-reduced states of cyt c554 in a new, rhombohedral crystal form have been solved by molecular replacement, at 1.6 Å and 1.8 Å resolution, respectively. Upon reduction, the conformation of the polypeptide chain changes between residues 175 and 179, which are adjacent to hemes III and IV. Cyt c554 displays conserved heme-packing motifs that are present in other heme-containing proteins. Comparisons to hydroxylamine oxidoreductase, the electron donor to cyt c554, and cytochrome c nitrite reductase, an enzyme involved in nitrite ammonification, reveal substantial structural similarity in the polypeptide chain surrounding the heme core environment. The structural determinants of these heme-packing motifs extend to the buried water molecules that hydrogen bond to the histidine ligands to the heme iron. In the original structure determination of a tetragonal crystal form, a cis peptide bond between His129 and Phe130 was identified that appeared to be stabilized by crystal contacts. In the rhombohedral crystal form used in the present high-resolution structure determination, this peptide bond adopts the trans conformation, but with disallowed angles of φ and ψ

Structure of the Escherichia coli Fumarate Reductase Respiratory Complex

The integral membrane protein fumarate reductase catalyzes the final step of anaerobic respiration when fumarate is the terminal electron acceptor. The homologous enzyme succinate dehydrogenase also plays a prominent role in cellular energetics as a member of the Krebs cycle and as complex II of the aerobic respiratory chain. Fumarate reductase consists of four subunits that contain a covalently linked flavin adenine dinucleotide, three different iron-sulfur clusters, and at least two quinones. The crystal structure of intact fumarate reductase has been solved at 3.3 angstrom resolution and demonstrates that the cofactors are arranged in a nearly linear manner from the membrane-bound quinone to the active site flavin. Although fumarate reductase is not associated with any proton-pumping function, the two quinones are positioned on opposite sides of the membrane in an arrangement similar to that of the Q-cycle organization observed for cytochrome bc_1

Analyzing Your Complexes: Structure of the Quinol-Fumarate Reductase Respiratory Complex

The integral membrane protein complex quinol-fumarate reductase catalyzes the terminal step of a major anaerobic respiratory pathway. The homologous enzyme succinate-quinone oxidoreductase participates in aerobic respiration both as complex II and as a member of the Krebs cycle. Last year, two structures of quinol-fumarate reductases were reported. These structures revealed the cofactor organization linking the fumarate and quinol sites, and showed a cofactor arrangement across the membrane that is suggestive of a possible energy coupling function

Cytochrome c_(554)

Cytochrome c_(554) (cyt c_(554)) is a tetra‐heme c‐type cytochrome that participates in the nitrification pathway of Nitrosomonas europaea. In this process, cyt c_(554) functions as the electron acceptor from the enzyme hydroxylamine oxidoreductase that catalyzes the oxidation of hydroxylamine to nitrite. Cyt c_(554) is a predominantly α‐helical protein with four covalently attached hemes. The four hemes are arranged in two pairs, such that the planes of the porphyrin rings are nearly parallel and overlapping at the edges. This type of heme‐stacking has been observed in two other nitrogen cycle proteins: hydroxylamine oxidoreductase and cytochrome c nitrite reductase, an enzyme that catalyzes the reduction of nitrite to ammonia. The relatively unusual spectral properties of cyt c_(554) may reflect interactions between these pairs of stacked hemes

Cytochrome c_(554)

Cytochrome c_(554) (cyt c_(554)) is a tetra‐heme c‐type cytochrome that participates in the nitrification pathway of Nitrosomonas europaea. In this process, cyt c_(554) functions as the electron acceptor from the enzyme hydroxylamine oxidoreductase that catalyzes the oxidation of hydroxylamine to nitrite. Cyt c_(554) is a predominantly α‐helical protein with four covalently attached hemes. The four hemes are arranged in two pairs, such that the planes of the porphyrin rings are nearly parallel and overlapping at the edges. This type of heme‐stacking has been observed in two other nitrogen cycle proteins: hydroxylamine oxidoreductase and cytochrome c nitrite reductase, an enzyme that catalyzes the reduction of nitrite to ammonia. The relatively unusual spectral properties of cyt c_(554) may reflect interactions between these pairs of stacked hemes