BBA - Bioenergetics (v.1777, #9)
Editorial Board (ii).
Regulatory interactions in the dimeric cytochrome bc 1 complex: The advantages of being a twin by Raul Covian; Bernard L. Trumpower (1079-1091).
The dimeric cytochrome bc 1 complex catalyzes the oxidation–reduction of quinol and quinone at sites located in opposite sides of the membrane in which it resides. We review the kinetics of electron transfer and inhibitor binding that reveal functional interactions between the quinol oxidation site at center P and quinone reduction site at center N in opposite monomers in conjunction with electron equilibration between the cytochrome b subunits of the dimer. A model for the mechanism of the bc 1 complex has emerged from these studies in which binding of ligands that mimic semiquinone at center N regulates half-of-the-sites reactivity at center P and binding of ligands that mimic catalytically competent binding of ubiquinol at center P regulates half-of-the-sites reactivity at center N. An additional feature of this model is that inhibition of quinol oxidation at the quinone reduction site is avoided by allowing catalysis in only one monomer at a time, which maximizes the number of redox acceptor centers available in cytochrome b for electrons coming from quinol oxidation reactions at center P and minimizes the leakage of electrons that would result in the generation of damaging oxygen radicals.
Keywords: bc 1 complex; Electron transfer; Quinone; Stigmatellin; Antimycin;
Mitochondrial fusion, fission and autophagy as a quality control axis: The bioenergetic view by Gilad Twig; Brigham Hyde; Orian S. Shirihai (1092-1097).
The mitochondrial life cycle consists of frequent fusion and fission events. Ample experimental and clinical data demonstrate that inhibition of either fusion or fission results in deterioration of mitochondrial bioenergetics. While fusion may benefit mitochondrial function by allowing the spreading of metabolites, protein and DNA throughout the network, the functional benefit of fission is not as intuitive. Remarkably, studies that track individual mitochondria through fusion and fission found that the two events are paired and that fusion triggers fission. On average each mitochondrion would go though ~ 5 fusion:fission cycles every hour. Measurement of Δψ m during single fusion and fission events demonstrates that fission may yield uneven daughter mitochondria where the depolarized daughter is less likely to become involved in a subsequent fusion and is more likely to be targeted by autophagy. Based on these observations we propose a mechanism by which the integration of mitochondrial fusion, fission and autophagy forms a quality maintenance mechanism. According to this hypothesis pairs of fusion and fission allow for the reorganization and sequestration of damaged mitochondrial components into daughter mitochondria that are segregated from the networking pool and then becoming eliminated by autophagy.
Keywords: Mitochondria; Membrane potential; Fusion; Fission; Autophagy;
Partial site-specific assignment of a uniformly 13C, 15N enriched membrane protein, light-harvesting complex 1 (LH1), by solid state NMR by Lei Huang; Ann E. McDermott (1098-1108).
Partial site-specific assignments are reported for the solid state NMR spectra of light-harvesting complex 1, a 160 kDa integral membrane protein. The assignments were derived from 600 MHz 15N–13CO–13Cα and 15N–13Cα–13CX correlation spectra, using uniformly 13C, 15N enriched hydrated material, in an intact and precipitated form. Sequential assignments were verified using characteristic 15N–13Cα–13Cβ side chain chemical shifts observed in 3D experiments. Tertiary contacts found in 2D DARR spectra of the selectively 13C enriched sample provided further confirmatory evidence for the assignments. The assignments include the region of the Histidine ligands binding the Bacteriochlorophyll chromophore. The chemical shifts of Cα and Cβ resonances indicated the presence of typical α-helical secondary structure, consistent with previous studies.
Keywords: Light-harvesting complex 1; Solid state NMR; Membrane protein; Sequential assignment;
Acceptor side effects on the electron transfer at cryogenic temperatures in intact photosystem II by Han Bao; Chunxi Zhang; Keisuke Kawakami; Yanan Ren; Jian-Ren Shen; Jingquan Zhao (1109-1115).
In intact PSII, both the secondary electron donor (TyrZ) and side-path electron donors (Car/ChlZ/Cyt b 559) can be oxidized by P680 + • at cryogenic temperatures. In this paper, the effects of acceptor side, especially the redox state of the non-heme iron, on the donor side electron transfer induced by visible light at cryogenic temperatures were studied by EPR spectroscopy. We found that the formation and decay of the S1TyrZ • EPR signal were independent of the treatment of K3Fe(CN)6, whereas formation and decay of the Car+ •/ChlZ + • EPR signal correlated with the reduction and recovery of the Fe3+ EPR signal of the non-heme iron in K3Fe(CN)6 pre-treated PSII, respectively. Based on the observed correlation between Car/ChlZ oxidation and Fe3+ reduction, the oxidation of non-heme iron by K3Fe(CN)6 at 0 °C was quantified, which showed that around 50–60% fractions of the reaction centers gave rise to the Fe3+ EPR signal. In addition, we found that the presence of phenyl-p-benzoquinone significantly enhanced the yield of TyrZ oxidation. These results indicate that the electron transfer at the donor side can be significantly modified by changes at the acceptor side, and indicate that two types of reaction centers are present in intact PSII, namely, one contains unoxidizable non-heme iron and another one contains oxidizable non-heme iron. TyrZ oxidation and side-path reaction occur separately in these two types of reaction centers, instead of competition with each other in the same reaction centers. In addition, our results show that the non-heme iron has different properties in active and inactive PSII. The oxidation of non-heme iron by K3Fe(CN)6 takes place only in inactive PSII, which implies that the Fe3+ state is probably not the intermediate species for the turnover of quinone reduction.
Keywords: Photosystem II; TyrZ; Side-path electron donor; Non-heme iron; EPR; Electron transfer;
Competitive inhibition of electron donation to photosystem 1 by metal-substituted plastocyanin by Hanna Jansson; Örjan Hansson (1116-1121).
The electron transfer from wild-type spinach plastocyanin (Pc) to photosystem 1 has been studied by flash-induced absorption changes at 830 nm. The decay kinetics of photo-oxidized P700 are drastically slower in the presence of Ag(I)-substituted Pc, while addition of Zn(II)-substituted Pc has a weaker effect. The metal-substituted forms of Pc act as competitive inhibitors of the reaction between normal, Cu-containing, Pc and P700. The inhibition constants obtained from an analysis of the kinetic data were 30 and 410 μM for Ag(I)- and Zn(II)-substituted Pc, respectively. When the Gly8Asp mutant form of Pc was used instead of the wild-type form, the corresponding values were found to be 77 and 442 μM. If the Ag- and Zn-derivatives can be considered as structural mimics of reduced and oxidized CuPc, respectively, our results imply that there is a redox-induced decrease in the affinity between Pc and photosystem 1 that follows the electron donation to P700. Our data also imply that the Gly8Asp mutation can diminish the magnitude of this change. The findings reported here are consistent with a reaction mechanism where the electron transfer in the complex between Pc and photosystem 1 is assumed to be reversible.
Keywords: Electron transfer; Photosystem 1; Plastocyanin; Redox-induced structural change;
Association of chlorophyll a/c 2 complexes to photosystem I and photosystem II in the cryptophyte Rhodomonas CS24 by Sami Kereïche; Roman Kouřil; Gert T. Oostergetel; Fabrizia Fusetti; Egbert J. Boekema; Alexander B. Doust; Chantal D. van der Weij-de Wit; Jan P. Dekker (1122-1128).
Photosynthetic supercomplexes from the cryptophyte Rhodomonas CS24 were isolated by a short detergent treatment of membranes from the cryptophyte Rhodomonas CS24 and studied by electron microscopy and low-temperature absorption and fluorescence spectroscopy. At least three different types of supercomplexes of photosystem I (PSI) monomers and peripheral Chl a/c 2 proteins were found. The most common complexes have Chl a/c 2 complexes at both sides of the PSI core monomer and have dimensions of about 17 × 24 nm. The peripheral antenna in these supercomplexes shows no obvious similarities in size and/or shape with that of the PSI–LHCI supercomplexes from the green plant Arabidopsis thaliana and the green alga Chlamydomonas reinhardtii, and may be comprised of about 6–8 monomers of Chl a/c 2 light-harvesting complexes. In addition, two different types of supercomplexes of photosystem II (PSII) dimers and peripheral Chl a/c 2 proteins were found. The detected complexes consist of a PSII core dimer and three or four monomeric Chl a/c 2 proteins on one side of the PSII core at positions that in the largest complex are similar to those of Lhcb5, a monomer of the S-trimer of LHCII, Lhcb4 and Lhcb6 in green plants.
Keywords: Photosystem I; Photosystem II; Antenna protein; Rhodomonas CS24; Electron microscopy; Cryptophytes;
Theoretical and computational analysis of the membrane potential generated by cytochrome c oxidase upon single electron injection into the enzyme by Ryogo Sugitani; Emile S. Medvedev; Alexei A. Stuchebrukhov (1129-1139).
We have developed theory and the computational scheme for the analysis of the kinetics of the membrane potential generated by cytochrome c oxidase upon single electron injection into the enzyme. The theory allows one to connect the charge motions inside the enzyme to the membrane potential observed in the experiments by using data from the “dielectric topography” map of the enzyme that we have created. The developed theory is applied for the analysis of the potentiometric data recently reported by the Wikström group [I. Belevich, D.A. Bloch, N. Belevich, M. Wikström and M.I. Verkhovsky, Exploring the proton pump mechanism of cytochrome c oxidase in real time, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 2685–2690] on the O to E transition in Paracoccus denitrificans oxidase. Our analysis suggests, that the electron transfer to the binuclear center is coupled to a proton transfer (proton loading) to a group just “above” the binuclear center of the enzyme, from which the pumped proton is subsequently expelled by the chemical proton arriving to the binuclear center. The identity of the pump site could not be determined with certainty, but could be localized to the group of residues His326 (His291 in bovine), propionates of heme a 3, Arg 473/474, and Trp164. The analysis also suggests that the dielectric distance from the P-side to Fe a is 0.4 or larger. The difficulties and pitfalls of quantitative interpretation of potentiometric data are discussed.
Keywords: Paracoccus denitrificans; Cytochrome c oxidase; Electron transfer; Proton translocation;
The Rieske/cytochrome b complex of Heliobacteria by A.L. Ducluzeau; E. Chenu; L. Capowiez; F. Baymann (1140-1146).
Heliobacteria have a Rieske/cytochrome b complex composed of a Rieske protein, a cytochrome b 6, a subunit IV and a di-heme cytochrome c. The overall structure of the complex seems close to the b 6 f complex from cyanobacteria and chloroplasts to the exception of the di-heme cytochrome. We show here by biochemical and biophysical studies that a heme c i is covalently attached to the Rieske/cytochrome b complex from Heliobacteria. We studied the EPR signature of this heme in two different species, Heliobacterium modesticaldum and Heliobacillus mobilis. In contrast to the case of b 6 f complex, a strong axial ligand to the heme is present, most probably a protonatable amino acid residue.
Keywords: bc complex; Heme c i; Heliobacteria; EPR; b 6 f complex;
Introduction of cytochrome b mutations in Saccharomyces cerevisiae by a method that allows selection for both functional and non-functional cytochrome b proteins by Martina G. Ding; Christine A. Butler; Scott A. Saracco; Thomas D. Fox; François Godard; Jean-Paul di Rago; Bernard L. Trumpower (1147-1156).
We have previously used inhibitors interacting with the Qn site of the yeast cytochrome bc 1 complex to obtain yeast strains with resistance-conferring mutations in cytochrome b as a means to investigate the effects of amino acid substitutions on Qn site enzymatic activity [M.G. Ding, J.-P. di Rago, B.L. Trumpower, Investigating the Qn site of the cytochrome bc1 complex in Saccharomyces cerevisiae with mutants resistant to ilicicolin H, a novel Qn site inhibitor, J. Biol. Chem. 281 (2006) 36036–36043.]. Although the screening produced various interesting cytochrome b mutations, it depends on the availability of inhibitors and can only reveal a very limited number of mutations. Furthermore, mutations leading to a respiratory deficient phenotype remain undetected. We therefore devised an approach where any type of mutation can be efficiently introduced in the cytochrome b gene. In this method ARG8, a gene that is normally encoded by nuclear DNA, replaces the naturally occurring mitochondrial cytochrome b gene, resulting in ARG8 expressed from the mitochondrial genome (ARG8 m ). Subsequently replacing ARG8 m with mutated versions of cytochrome b results in arginine auxotrophy. Respiratory competent cytochrome b mutants can be selected directly by virtue of their ability to restore growth on non-fermentable substrates. If the mutated cytochrome b is non-functional, the presence of the COX2 respiratory gene marker on the mitochondrial transforming plasmid enables screening for cytochrome b mutants with a stringent respiratory deficiency (mit−). With this system, we created eight different yeast strains containing point mutations at three different codons in cytochrome b affecting center N. In addition, we created three point mutations affecting arginine 79 in center P. This is the first time mutations have been created for three of the loci presented here, and nine of the resulting mutants have never been described before.
Keywords: Cytochrome b; Mutations; Biolistic transformation; Yeast; Cytochrome bc1 complex;
Structural insights into the modulation of the redox properties of two Geobacter sulfurreducens homologous triheme cytochromes by Leonor Morgado; Marta Bruix; Valerie Orshonsky; Yuri Y. Londer; Norma E.C. Duke; Xiaojing Yang; P. Raj Pokkuluri; Marianne Schiffer; Carlos A. Salgueiro (1157-1165).
The redox properties of a periplasmic triheme cytochrome, PpcB from Geobacter sulfurreducens, were studied by NMR and visible spectroscopy. The structure of PpcB was determined by X-ray diffraction. PpcB is homologous to PpcA (77% sequence identity), which mediates cytoplasmic electron transfer to extracellular acceptors and is crucial in the bioenergetic metabolism of Geobacter spp. The heme core structure of PpcB in solution, probed by 2D-NMR, was compared to that of PpcA. The results showed that the heme core structures of PpcB and PpcA in solution are similar, in contrast to their crystal structures where the heme cores of the two proteins differ from each other. NMR redox titrations were carried out for both proteins and the order of oxidation of the heme groups was determined. The microscopic properties of PpcB and PpcA redox centers showed important differences: (i) the order in which hemes become oxidized is III–I–IV for PpcB, as opposed to I–IV–III for PpcA; (ii) the redox-Bohr effect is also different in the two proteins. The different redox features observed between PpcB and PpcA suggest that each protein uniquely modulates the properties of their co-factors to assure effectiveness in their respective metabolic pathways. The origins of the observed differences are discussed.
Keywords: Geobacter; Multiheme cytochrome; Electron transfer; Redox-Bohr effect;
Conserved lysine residues of the membrane subunit NuoM are involved in energy conversion by the proton-pumping NADH:ubiquinone oxidoreductase (Complex I) by Liliya Euro; Galina Belevich; Michael I. Verkhovsky; Mårten Wikström; Marina Verkhovskaya (1166-1172).
Analysis of the amino acid sequences of subunits NuoM and NuoN in the membrane domain of Complex I revealed a clear common pattern, including two lysines that are predicted to be located within the membrane, and which are important for quinone reductase activity. Site-directed mutations of the amino acid residues E144, K234, K265 and W243 in this pattern were introduced into the chromosomal gene nuoM of Escherichia coli Complex I. The activity of mutated Complex I was studied in both membranes and in purified Complex I. The quinone reductase activity was practically lost in K234A, K234R and E144A, decreased in W243A and K265A but unchanged in E144D. Complex I from all these mutants contained 1 mol tightly bound ubiquinone per mol FMN like wild type enzyme. The mutant enzymes E144D, W243A and K265A had wild type sensitivity to rolliniastatin and complete proton-pumping efficiency of Complex I. Remarkably, the subunits NuoL and NuoH in the membrane domain also appear to contain conserved lysine residues in transmembrane helices, which may give a clue of the mechanism of proton translocation. A tentative principle of proton translocation by Complex I is suggested based on electrostatic interactions of lysines in the membrane subunits.
Keywords: Complex I; NuoM subunit; Proton-pumping mechanism;
Competition between linear and cyclic electron flow in plants deficient in Photosystem I by Simon Hald; Mathias Pribil; Dario Leister; Patrick Gallois; Giles N. Johnson (1173-1183).
Photosynthetic electron transport can involve either a linear flow from water to NADP, via Photosystems (PS) II and I or a cyclic flow just involving PSI. Little is known about factors regulating the relative flow through each of these pathways. We have examined photosynthetic electron transport through each system in plants of Arabidopsis thaliana in which either the PSI-D1 or PSI-E1 subunits of PSI have been knocked out. In both cases, this results in an imbalance in the turnover of PSI and PSII, such that PSII electron transport is limited by PSI turnover. Phosphorylation of light-harvesting complex II (LHCII) and its migration to PSI is enhanced but only partially reversible and not sufficient to balance photosystem turnover. In spite of this, cyclic electron flow is able to compete efficiently with PSI across a range of conditions. In dark-adapted leaves, the efficiency of cyclic relative to linear flow induced by far-red light is increased, implying that the limiting step of cyclic flow lies in the re-injection of electrons into the electron transport chain. Illumination of leaves with white light resulted in transient induction of a significant non-photochemical quenching in knockout plants which is probably high energy state quenching induced by cyclic electron flow. At high light and at low CO2, non-photochemical quenching was greater in the knockout plants than in the wildtype. Comparison of PSI and PSII turnover under such conditions suggested that this is generated by cyclic electron flow around PSI. We conclude that, when the concentration of PSI is limiting, cyclic electron flow is still able to compete effectively with linear flow to maintain a high ΔpH to regulate photosynthesis.
Keywords: Linear electron flow; Cyclic electron flow; Photosystem I; Photosystem II; PSI-D1; PSI-E1; Arabidopsis thaliana;
Role of phosphatidylglycerol in the function and assembly of Photosystem II reaction center, studied in a cdsA-inactivated PAL mutant strain of Synechocystis sp. PCC6803 that lacks phycobilisomes by Hajnalka Laczkó-Dobos; Bettina Ughy; Szilvia Z. Tóth; Josef Komenda; Ottó Zsiros; Ildikó Domonkos; Árpád Párducz; Balázs Bogos; Masayuki Komura; Shigeru Itoh; Zoltán Gombos (1184-1194).
To analyze the role of phosphatidylglycerol (PG) in photosynthetic membranes of cyanobacteria we used two mutants of Synechocystis sp. PCC6803: the PAL mutant which has no phycobilisomes and shows a high PSII/PSI ratio, and a mutant derived from it by inactivating its cdsA gene encoding cytidine 5'-diphosphate diacylglycerol synthase, a key enzyme in PG synthesis. In a medium supplemented with PG the PAL/ΔcdsA mutant cells grew photoautotrophically. Depletion of PG in the medium resulted (a) in an arrest of cell growth and division, (b) in a slowdown of electron transfer from the acceptor QA to QB in PSII and (c) in a modification of chlorophyll fluorescence curve. The depletion of PG affected neither the redox levels of QA nor the S2 state of the oxygen-evolving manganese complex, as indicated by thermoluminescence studies. Two-dimensional PAGE showed that in the absence of PG (a) the PSII dimer was decomposed into monomers, and (b) the CP43 protein was detached from a major part of the PSII core complex. [35S]-methionine labeling confirmed that PG depletion did not block de novo synthesis of the PSII proteins. We conclude that PG is required for the binding of CP43 within the PSII core complex.
Keywords: Synechocystis sp. PCC6803; Phosphatidylglycerol; Photosystem II; Thermoluminescence; OJIP transient; Cell division;
Stepping behavior of two-headed kinesin motors by Ping Xie (1195-1202).
The stepping behavior of the dimeric kinesin is studied by using our model based on previous biochemical, X-ray crystallography and cryo-electron microscopy studies. It is shown that, when a Pi is released from the trailing head, a forward step is made under a backward load smaller than the stall force; while when a Pi is released from the leading head, no stepping is made under a forward load or no load, and a backward step is made under a backward load. The forward stepping time, i.e., the time from the release of Pi in the trailing head to the binding of the ADP head to next binding site, is much smaller than the dwell time even under the backward load near the stall force. Thus the movement velocity of the kinesin dimer can be considered to be only dependent on ATPase rates of the two heads. The duration of the rising phase, i.e., the actual time taken by the ADP head to transit from the trailing to leading positions, is on the time scale of microseconds under any backward load smaller than the stall force. This is consistent with available experimental results.
Keywords: Kinesin-1; Molecular motor; Mechanochemstry; Model;
Direct and mediated electron transfer between intact succinate:quinone oxidoreductase from Bacillus subtilis and a surface modified gold electrode reveals redox state-dependent conformational changes by Andreas Christenson; Tobias Gustavsson; Lo Gorton; Cecilia Hägerhäll (1203-1210).
Succinate:quinone oxidoreductase (SQR) from Bacillus subtilis consists of two hydrophilic protein subunits comprising succinate dehydrogenase, and a di-heme membrane anchor protein harboring two putative quinone binding sites, Qp and Qd. In this work we have used spectroelectrochemistry to study the electronic communication between purified SQR and a surface modified gold capillary electrode. In the presence of two soluble quinone mediators the midpoint potentials of both hemes were revealed essentially as previously determined by conventional redox titration (heme bH, E m = + 65 mV, heme bL, E m = − 95 mV). In the absence of mediators the enzyme still communicated with the electrode, albeit with a reproducible hysteresis, resulting in the reduction of both hemes occurring approximately at the midpoint potential of heme bL, and with a pronounced delay of reoxidation. When the specific inhibitor 2-n-heptyl-4 hydroxyquinoline N-oxide (HQNO), which binds to Qd in B. subtilis SQR, was added together with the two quinone mediators, rapid reductive titration was still possible which can be envisioned as an electron transfer occurring via the HQNO insensitive Qp site. In contrast, the subsequent oxidative titration was severely hampered in the presence of HQNO, in fact it completely resembled the unmediated reaction. If mediators communicate with Qp or Qd, either event is followed by very rapid electron redistribution within the enzyme. Taken together, this strongly suggests that the accessibility of Qp depended on the redox state of the hemes. When both hemes were reduced, and Qd was blocked by HQNO, quinone-mediated communication via the Qp site was no longer possible, revealing a redox-dependent conformational change in the membrane anchor domain.
Keywords: SQR; QFR; SdhC; Cytochrome b; Quinone; Mercaptohexanol; Spectroelectrochemistry; Gold capillary;
Parallel electron donation pathways to cytochrome c z in the type I homodimeric photosynthetic reaction center complex of Chlorobium tepidum by Yusuke Tsukatani; Chihiro Azai; Toru Kondo; Shigeru Itoh; Hirozo Oh-oka (1211-1217).
We studied the regulation mechanism of electron donations from menaquinol:cytochrome c oxidoreductase and cytochrome c-554 to the type I homodimeric photosynthetic reaction center complex of the green sulfur bacterium Chlorobium tepidum. We measured flash-induced absorption changes of multiple cytochromes in the membranes prepared from a mutant devoid of cytochrome c-554 or in the reconstituted membranes by exogenously adding cytochrome c-555 purified from Chlorobium limicola. The results indicated that the photo-oxidized cytochrome c z bound to the reaction center was rereduced rapidly by cytochrome c-555 as well as by the menaquinol:cytochrome c oxidoreductase and that cytochrome c-555 did not function as a shuttle-like electron carrier between the menaquinol:cytochrome c oxidoreductase and cytochrome c z. It was also shown that the rereduction rate of cytochrome c z by cytochrome c-555 was as high as that by the menaquinol:cytochrome c oxidoreductase. The two electron-transfer pathways linked to sulfur metabolisms seem to function independently to donate electrons to the reaction center.
Keywords: Cytochrome c z; Cytochrome c-554; Green sulfur bacteria; Menaquinol:cytochrome c oxidoreductase; Reaction center; Sulfur oxidation;
His92 and His110 selectively affect different heme centers of adrenal cytochrome b561 by Wen Liu; Corina E. Rogge; Giordano F.Z. da Silva; Vladimir P. Shinkarev; Ah-Lim Tsai; Yury Kamensky; Graham Palmer; Richard J. Kulmacz (1218-1228).
Adrenal cytochrome b561 (cyt b561 ), a transmembrane protein that shuttles reducing equivalents derived from ascorbate, has two heme centers with distinct spectroscopic signals and reactivity towards ascorbate. The His54/His122 and His88/His161 pairs furnish axial ligands for the hemes, but additional amino acid residues contributing to the heme centers have not been identified. A computational model of human cyt b561 (Bashtovyy, D., Berczi, A., Asard, H., and Pali, T. (2003) Protoplasma 221, 31–40) predicts that His92 is near the His88/His161 heme and that His110 abuts the His54/His122 heme. We tested these predictions by analyzing the effects of mutations at His92 or His110 on the spectroscopic and functional properties. Wild type cytochrome and mutants with substitutions in other histidine residues or in Asn78 were used for comparison. The largest lineshape changes in the optical absorbance spectrum of the high-potential (bH) peak were seen with mutation of His92; the largest changes in the low-potential (bL) peak lineshape were observed with mutation of His110. In the EPR spectra, mutation of His92 shifted the position of the g = 3.1 signal (bH) but not the g = 3.7 signal (bL). In reductive titrations with ascorbate, mutations in His92 produced the largest increase in the midpoint for the bH transition; mutations in His110 produced the largest decreases in ΔA 561 for the bL transition. These results indicate that His92 can be considered part of the bH heme center, and His110 part of the bL heme center, in adrenal cyt b561 .
Keywords: Adrenal cytochrome b561 ; Heme center; Histidine residue;