Q-Band resonance Raman investigation of turnip cytochrome f and Rhodobacter capsulatus cytochrome c1.

The results of a comprehensive Q-band resonance Raman investigation of cytochrome c1 and cytochrome f subunits of bc1 and b6f complexes are presented. Q-band excitation provides a particularly effective probe of the local heme environments of these species. The effects of protein conformation (particularly axial ligation) on heme structure and function were further investigated by comparison of spectra obtained from native subunits to those of a site directed c1 mutant (M183L) and various pH-dependent species of horse heart cytochrome c. In general, all species examined displayed variability in their axial amino acid ligation that suggests a good deal of flexibility in their hemepocket conformations. Surprisingly, the large scale protein rearrangements that accompany axial ligand replacement have little or no effect on macrocycle geometry in these species. This indicates the identity and/or conformation of the peptide linkage between the two cysteines that are covalently linked to the heme periphery may determine heme geometry.

Axial heme ligation in the cytochrome bc1 complexes of mitochondrial and photosynthetic membranes. A near-infrared magnetic circular dichroism and electron paramagnetic resonance study.

The combination of EPR and low-temperature near-IR magnetic circular dichroism spectroscopies have been used to investigate the axial ligation of the cytochromes in the cytochrome bc1 complexes from bovine heart mitochondria, Rhodobacter capsulatus, Rhodobacter sphaeroides, and Rhodospirillum rubrum, and the purified cytochromes c1 from bovine heart mitochondria, Rb. capsulatus and Rb. sphaeroides. The possibility of axial ligation of cytochrome c1 by the amino terminus of the polypeptide was also assessed by acetylating the N-terminus of Rb. capsulatus cytochrome c1 and comparing the properties of the acetylated and unmodified samples. The results are consistent with bis-histidine axial ligation for the high- and low-potential b-type cytochromes and histidine/methionine axial ligation for the c1-type cytochrome in the intact cytochrome bc1 complexes. Purified samples of cytochrome c1 are mixtures of two forms, one with histidine/methionine and the other with bis-histidine axial ligation. The form with bis-histidine axial ligation is also assembled in the M183L mutant of the Rb. capsulatus cyt bc1 complex in which the methionine residue coordinating cyt c1 is replaced by a leucine. The bis-histidine form appears to be an artifact of dissociation of cytochrome c1 from the cytochrome bc1 complex and is greatly enhanced particularly in the bacterial cytochromes c1 by sample handling and the addition of 50% (v/v) ethylene glycol or glycerol.

Effects of cold storage on the subsequent structure and function of microvenous autografts.

A study was undertaken to evaluate the structure and patency of cold stored rabbit femoral veins following reinsertion as autografts for 3 weeks. The periods of cold ischaemic storage were 1, 2, 3 and 4 weeks (n = 10/gp) and 6 and 10 weeks (n = 6/gp). All rabbits were subject to 3 operations under general anaesthesia. In the first, a 4 cm segment of left femoral vein was harvested and stored at 4 degrees C for the specified ischaemic interval. Following storage the graft was microsurgically reinserted at a second operation into the right femoral artery of the donor rabbit. Three weeks later, graft patency was assessed by surgical exploration and the graft processed for light and electron microscopy. Patency rates remained over 80% after 3 weeks in all groups except the 10 week storage group where only 1 of 6 (17%) grafts was patent at 3 weeks. In all groups normal vein structure was absent, being replaced by thin walled necrotic areas or by neointimal ingrowth. The excellent patency rates achieved indicate it is possible to cold preserve extra lengths of vein grafts harvested at initial operation for reuse should regrafting be necessary.

A look at adolescent treatment in a time of change.

Multiple changes in the authors' inpatient adolescent unit--including unit mergers, increased patient acuity, and shorter length of stay--created the need and opportunity to implement a new Behavioral Milieu Program with two significant components: A positive Behavioral Points System and 15 nurse-led groups. The authors describe this new program, which provides a safe and effective treatment modality and promotes each adolescent's sense of responsibility for his own behavior and life. Dramatic outcomes include decreased use of mechanical restraints by 56% in the first three months of implementation and by 82% in the second three months. This program has applications for inpatient units, community residences, and schools, and can even be adapted for home use by parents.

Rhodobacter capsulatus contains a novel cb-type cytochrome c oxidase without a CuA center.

The facultative phototrophic bacterium Rhodobacter capsulatus is capable of growth in a wide range of environmental conditions using a highly branched electron-transfer chain. During respiratory growth of this organism reducing equivalents are conveyed to oxygen via two terminal oxidases, previously called "cyt b410" (cytochrome c oxidase) and "cyt b260" (quinol oxidase). The cytochrome c oxidase was purified to homogeneity from a semiaerobically grown R. capsulatus strain. The purified enzyme consumes oxygen at a rate of 600 s-1, oxidizes reduced equine cyt c and R. capsulatus cyt c2, and has high sensitivity to cyanide. The complex is composed of three major polypeptides of apparent molecular masses 45, 32, and 28 kDa on SDS-PAGE. The 32- and 28-kDa proteins also stain with tetramethylbenzidine, indicating that they are c-type cytochromes. Partial amino acid sequences obtained from each of the subunits reveal significant homology to the fixN, fixO, and fixP gene products of Bradyrhizobium japonicum and Rhizobium meliloti. The reduced enzyme has an optical absorption spectrum with distinct features near 550 and 560 nm and an asymmetric Soret band centered at 418 nm, indicating the presence of both c- and b-type cytochromes. Two electrochemically distinct cyt c are apparent, with redox midpoint potentials (Em7) of 265 and 320 mV, while the low-spin cyt b has an Em7 value of 385 mV. The enzyme binds carbon monoxide, and the CO difference spectrum indicates that CO binds to a high-spin cyt b. Pyridine hemochrome and HPLC analyses suggest that the complex contains 1 mol of heme C to 1 mol of protoheme and that neither heme O nor heme A is present.(ABSTRACT TRUNCATED AT 250 WORDS)

Requirement of histidine 217 for ubiquinone reductase activity (Qi site) in the cytochrome bc1 complex.

Folding models suggest that the highly conserved histidine 217 of the cytochrome b subunit from the cytochrome bc1 complex is close to the quinone reductase (Qi) site. This histidine (bH217) in the cytochrome b polypeptide of the photosynthetic bacterium Rhodobacter capsulatus has been replaced with three other residues, aspartate (D), arginine (R), and leucine (L). bH217D and bH217R are able to grow photoheterotrophically and contain active cytochrome bc1 complexes (60% of wild-type activity), whereas the bH217L mutant is photosynthetically incompetent and contains a cytochrome bc1 complex that has only 10% of the wild-type activity. Single-turnover flash-activated electron transfer experiments show that cytochrome bH is reduced via the Qo site with near native rates in the mutant strains but that electron transfer between cytochrome bH and quinone bound at the Qi site is greatly slowed. These results are consistent with redox midpoint potential (Em) measurements of the cytochrome b subunit hemes and the Qi site quinone. The Em values of cyt bL and bH are approximately the same in the mutants and wild type, although the mutant strains have a larger relative concentration of what may be the high-potential form of cytochrome bH, called cytochrome b150. However, the redox properties of the semiquinone at the Qi site are altered significantly. The Qi site semiquinone stability constant of bH217R is 10 times higher than in the wild type, while in the other two strains (bH217D and bH217L) the stability constant is much lower than in the wild type. Thus H217 appears to have major effects on the redox properties of the quinone bound at the Qi site. These data are incorporated into a suggestion that H217 forms part of the binding pocket of the Qi site in a manner reminiscent of the interaction between quinone bound at the Qb site and H190 of the L subunit of the bacterial photosynthetic reaction center.

The functional and structural effects of hypothermic storage on ischaemic arterial grafts.

The effects of hypothermic ischaemia on blood vessels are unknown. This study aimed to determine the 3 week patency rate and the pathology of 9 experimental groups of hypothermically stored ischaemic arteries and one control group in a rabbit femoral artery model. Ischaemia times were 0 h, 24 h, 1, 2, 4, 6, 8 and 10 weeks (Groups 1-8). Patency was over 80% in all groups after 3 weeks reinsertion. Following reinsertion control grafts maintained normal arterial structure, but cellular degeneration had occurred in all ischaemic grafts and appeared complete after 4 weeks ischaemia. The graft connective tissue framework frequently remained intact. Repair was evident in central graft regions after 2 weeks ischaemia and 3 weeks reinsertion, but occurred only adjacent to the anastomosis in 4-10 week ischaemic arteries. Four week ischaemic arteries (Groups 9 and 10) reinserted for 6 and 12 weeks respectively exhibited near complete repair but patency dropped to 60% in the 12 week group.

Mutagenesis of methionine-183 drastically affects the physicochemical properties of cytochrome c1 of the bc1 complex of Rhodobacter capsulatus.

Site-directed mutagenesis was used to investigate which of the highly conserved methionine residues (M183 and M205) provides the sixth axial ligand to the heme Fe in the cyt c1 subunit of the bc1 complex from the bacterium Rhodobacter capsulatus. These residues were changed to leucine (cM183L) and valine (cM205V). Two additional mutants were constructed, 1 in which a stop codon was inserted at M205 (cM205*) and the second in which 127 amino acids were deleted between the signal sequence and the putative C-terminal transmembrane alpha-helix (c delta SfuI). Only cM205V grew photosynthetically, and membranes isolated from this strain catalyzed quinol-dependent reduction of cyt c in amounts similar to that in a wild-type strain. Even though cM183L could not grow photosynthetically, it contained all the appropriate polypeptides and cofactors of the bc1 complex, as shown by SDS-PAGE and optical difference spectroscopy of intact membrane particles. Neither of the two deletion mutants contained a stable complex. Flash absorption spectroscopy using chromatophores showed no cytochrome c rereduction after oxidation by the reaction center in cM183L. The bc1 complex from each strain was isolated and characterized. Oxidation reduction midpoint potential titrations revealed that cyt c1 from cM183L had a dramatically shifted Em value (delta Em = -390 mV) compared with wild type and cM205V. While the optical absorption spectrum of cyt c1 from cM183L suggested that the c-type heme was low-spin, nonetheless it was able to react with the exogenous ligand carbon monoxide. The overall data support that M183, and not M205, is the sixth ligand to the heme Fe of cyt c1 of the bc1 complex.

Interaction between thromboxane and free radical mechanisms in experimental ischaemic rabbit skin flaps.

The possible relationship between increased blood levels of thromboxane (TXA2) and tissue levels of free radicals during ischaemia was investigated. Rabbit epigastric skin flaps were subjected to 4 h of body temperature ischaemia, then infused with either the TXA2 synthetase inhibitor UK-38,485, the free radical scavenger superoxide dismutase (SOD), or both immediately prior to reperfusion. After 30 min of reperfusion, increases in the tissue levels of xanthine oxidase (XO) and malonyldialdehyde (MDA), both of which are indices of free radical generation and decreases in the tissue levels of SOD were found. SOD treatment completely restored XO, MDA and SOD levels to normal, whereas UK-38,485 only partially improved all three parameters. None of these changes was statistically significant. Effluent blood thromboxane B2 (TXB2) levels from the flap increased significantly (P less than 0.01) after ischaemia and were reduced significantly by both UK-38,485 and SOD (P less than 0.05). Combined UK-38,485 and SOD treatment was no better than treatment with either agent alone. ATP levels and oedema, which decreased and increased respectively due to ischaemia, were not significantly altered by drug infusion. These results suggest that free radical damage may be related to TXA2-generated thrombosis in ischaemia/reperfusion injury.