The crystal structure of the cyanobacterial cytochrome b 6 f complex has previously been solved to 3.0-Å resolution using the thermophilic Mastigocladus laminosus whose genome has not been sequenced. Several unicellular cyanobacteria, whose genomes have been sequenced and are tractable for mutagenesis, do not yield b 6 f complex in an intact dimeric state with significant electron transport activity. The genome of Nostoc sp. PCC 7120 has been sequenced and is closer phylogenetically to M. laminosus than are unicellular cyanobacteria. The amino acid sequences of the large core subunits and four small peripheral subunits of Nostoc are 88 and 80% identical to those in the M. laminosus b 6 f complex. Purified b 6 f complex from Nostoc has a stable dimeric structure, eight subunits with masses similar to those of M. laminosus, and comparable electron transport activity. The crystal structure of the native b 6 f complex, determined to a resolution of 3.0 Å (PDB id: 2ZT9), is almost identical to that of M. laminosus. Two unique aspects of the Nostoc complex are: (i) a dominant conformation of heme b p that is rotated 180°a bout the ␣-and ␥-meso carbon axis relative to the orientation in the M. laminosus complex and (ii) acetylation of the Rieske iron-sulfur protein (PetC) at the N terminus, a post-translational modification unprecedented in cyanobacterial membrane and electron transport proteins, and in polypeptides of cytochrome bc complexes from any source. The high spin electronic character of the unique heme c n is similar to that previously found in the b 6 f complex from other sources.
Extradiol catecholic dioxygenases catalyze the cleavage of the aromatic ring of the substrate with incorporation of both oxygen atoms from O 2 . 1 This reaction is a key step in the ability of Nature to reclaim large quantities of carbon sequestered in aromatic compounds. The active sites of these enzymes contain either Mn or Fe, coordinated by a 2-His-1-carboxylate facial triad, which is a common motif of nonheme Fe(II)-containing enzymes that activate dioxygen. 2 Homoprotocatechuate 2,3-dioxygenase from either Brevi-bacterium fuscum or Arthrobacter globiformis catalyzes the ring opening of homoprotocatechuate (HPCA) and contains Fe(II) (FeHPCD) or Mn(II) (MnMndD), respectively, as the native metals. Structures of FeHPCD from crystals that had been incubated with the substrate analogue 4-nitrocatechol and then exposed to low concentrations of O 2 identified three intermediate complexes in the catalytic cycle: semiquinone substrate radical-Fe superoxo (E-SQ), Fe-alkylperoxo (E-AP), and the Fesemialdehyde ring-opened product (EP). 3 Bond length analysis suggests that the iron remains ferrous in each species. A mechanistic proposal developed from these intermediates and earlier studies suggests that electron transfer from the substrate to O 2 , via the metal to form the E-SQ intermediate, results in simultaneous activation of both substrate and oxygen. Recent work has shown that FeHPCD and MnMndD can each be prepared with the nonphysiological metal in the active site (MnHPCD and FeMndD) and that all four forms have approximately the same catalytic parameters. 4 The fact that FeHPCD and MnHPCD also have superimposable structures suggests that the bound metals retain their inherent ~0.7 V difference in redox potential, leading to the proposal that the metal does not undergo a redox state change in the reaction cycle.Here we explore this question through direct electron paramagnetic resonance (EPR) detection and quantitative analysis of the metal oxidation states of MnHPCD as it turns over the natural substrate, HPCA. MnHPCD was chosen for study because it is available in a pure state, it is EPR active, and its full length form is structurally characterized. Four distinct Mn species are observed, two of which are short-lived intermediates. Our advances in EPR simulation software 5, 6 allow characterization of the electronic environment of all Mn species and the determination of concentrations of all species during turnover. Based on this analysis, a lowconcentration intermediate appearing immediately after O 2 addition is found to contain Mn (III) coupled to a radical. Thus, it is possible that the rapid electron transfer to form the reactive Figure 1. The substrate-free enzyme (E, Figure 1A) shows a six-line hyperfine pattern centered at g = 2.00, with splitting of a = 89 G, and a broad feature at g = 2.52. The hyperfine constant is indicative of a Mn(II) species, and the simulation of this spectrum (spectrum A) is calculated for a single protein-bound Mn(II) species using the parameters given in the Figure 1 cap...
Cytochrome b6f of oxygenic photosynthesis was studied using multifrequency, multimode EPR Spectroscopy. Frequency dependent signals above g = 4.3, and the observation of parallel-mode signals, are indicative of spin interactions in the complex. We demonstrate the presence of an exchange interaction between the unique high-spin heme cn and a nearby low-spin heme bn, and show that a quinone analog NQNO binds at or near to heme cn. The two hemes remain spin coupled upon the binding of NQNO, though strength of interaction decreases significantly. The electronic coupling implies that the heme bn/cn pair could function as a unit to facilitate 2-electron reduction of plastoquionone without generation of an energetically unfavorable semiquinone intermediate.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.