The structure of cytochromeb562fromEscherichia colihas been refined at 1.4 Å resolution against X-ray data collected on a Picker four-circle diffractometer. The triclinic unit cell parameters area=33.68 Å,b=50.48 Å,c=32.67 Å, α=102.51°, β=86.56° and γ=107.01° and there are two molecules in the asymmetric unit. A total of 138 cycles of restrained crystallographic refinement using the program PROLSQ were augmented at intermediate stages by two cycles of simulated annealing refinement using X-PLOR. The final crystallographicR-factor is 16.4% for data in the resolution range 6.0 Å to 1.4 for a model containing 1650 protein atoms, 86 heme atoms, 165 water molecules and four sulfate anions. The root-mean-square deviations from ideal bond lengths and angles are 0.012 Å and 2.0°, respectively. Each molecule consists of a bundle of four α-helices arranged in a simple up-dooown-up-down manner with a non-covalently bound heme group inserted between the first and fourth helices. In addition, there is a very short 310helix in the 15-residue loop connecting the first and second pairs of helices. The two independent molecules show r.m.s. differences of 0.30 Å for main-chain atoms and 0.88 Å for all atoms. A detailed comparison with the structurally similar cytochromec° fromRhodospppirulum molishianumis presented. In addition, the titration behavior of cytochromeb562in solution is discussed in terms of its molecular structure.

The structure of flavocytochrome b2 from baker's yeast was solved at 3.0-A resolution by the multiple isomorphous replacement method combined with solvent leveling procedures, using data collected from an area detector. The tetramer of Mr 230,000 has 4-fold symmetry. Each subunit contains a cytochrome domain consisting of the first 100 residues, a flavin-binding domain containing the next 386 residues, and an extended C-terminal tail of 25 residues. The cytochrome domain closely resembles microsomal cytochrome b5, whereas the flavin-binding domain contains a parallel beta 8/alpha 8 barrel motif similar to glycolate oxidase and trimethylamine dehydrogenase. Two of the four cytochrome domains are disordered in the crystals. The flavin ring and heme group are separated by about 16 A between their centers, and their planes are inclined by about 17 degrees to each other.

The structure of flavocytochrome b2 from baker's yeast was solved at 3.0-A resolution by the multiple isomorphous replacement method combined with solvent leveling procedures, using data collected from an area detector. The tetramer of Mr 230,000 has 4-fold symmetry. Each subunit contains a cytochrome domain consisting of the first 100 residues, a flavin-binding domain containing the next 386 residues, and an extended C-terminal tail of 25 residues. The cytochrome domain closely resembles microsomal cytochrome b5, whereas the flavin-binding domain contains a parallel beta 8/alpha 8 barrel motif similar to glycolate oxidase and trimethylamine dehydrogenase. Two of the four cytochrome domains are disordered in the crystals. The flavin ring and heme group are separated by about 16 A between their centers, and their planes are inclined by about 17 degrees to each other.

The ROCKS crystallographic computing system is a general complete integrated and easy-to-use system of programs for macromolecular structure determination. However, it has heretofore been available only on IBM 360 or 370 main-frame computers. This note reports the successful adaptation of the ROCKS system to the DEC VAX series of superminicomputers.

The atomic models of the cytochrome b562 and cytochrome c' monomers have been compared. When the respective heme groups are superimposed, the four alpha-helices of each nearly coincide. Four aromatic side chains, including the heme ligands, and a methionine occur in spatially equivalent positions in contact with the heme groups. This structural evidence suggests that the two cytochrome families may have diverged from a common molecular ancestor.

The amino acid sequence of cytochrome b562 has been redetermined and fitted to a 2.5 Å electron density map. A combination of cyanogen bromide fragmentation, proteolytic cleavages and manual and automatic sequencing was used. The model was built on an MMS-X molecular graphics system by interactively fitting the model to the electron density.

The structure of cytochrome b562 from Escherichia coli has been determined at 2.5 A resolution by x-ray diffraction methods. Protein phases were computed by the single isomorphous replacement method with anomalous scattering measurements from the native and uranyl acetate-substituted crystals. The electron density was averaged about the noncrystallographic 2-fold axis relating 2 molecules in the triclinic unit cell. The protein consists of four nearly parallel alpha helices and represents a new class of cytochrome structure. The heme group is inserted between the helices near one end of the molecule with one heme face partially exposed to solvent. The two heme ligands are histidine and methionine. The 2 phenylalanines are packed internally near the heme group, and the 2 tyrosines are on the surface, also near the heme group. The folding of the protein resembles that of hemerythrin and tobacco mosaic virus protein and shows a different topology from that of cytochrome b5, cytochrome c, or the globins.

A rotation-function study of flavocytochrome b2 has been carried out using X-ray data from crystals which contain one tetramer of 230 000 Dalton per asymmetric unit. The function computed from 10-5.5 Å resolution data clearly shows the orientation of the molecular 222 symmetry axes. The presence of these symmetry elements is consistent with previous structural and biochemical studies of the molecule.

X-RAY crystallography and nuclear magnetic resonance (NMR) are the two most powerful physical techniques for studying macromolecular structure. They are often used to complement one another in the study of a particular molecule. Crystallographic results have been of tremendous help in the NMR peak assignments which are subsequently used to obtain structural information in solution1,2. We report the combined use of both X-ray crystallography and NMR to identify MET-7 and HIS-106 as the two ligands of the haem iron of cytochrome b562.

Three crystalline forms of cytochrome b562 from Escherichia coli have been grown. One form, suitable for a three-dimensional structure investigation, is triclinic, containing two molecules per cell, with the following parameters: a = 33·71 Å, b = 50·51 Å, c = 32·71 Å, α = 102.62°, β = 86.67° and γ = 107.03°. X-ray data from the native crystals have been collected to 2.8 Å resolution and a search for suitable heavy atom derivatives has begun.