The nucleo-, phospho- and matrix protein of measles virus were localized at high resolution within infected cells by use of post-embedding immunogold labelling techniques. In general, labelling with monospecific antibodies as well as with a polyvalent rabbit anti-measles hyperimmune antiserum revealed measles virus polypeptides to be distributed non-randomly within infected cells with the label largely confined to specific sites, namely inclusions of nucleocapsids and assembled virus structures at the plasma membrane. Immunogold double labelling indicated that the phosphoprotein strictly co-localized with the nucleoprotein in cytoplasmic inclusions of nucleocapsids and in budding virions, whereas intranuclear inclusions of nucleocapsids were devoid of phosphoprotein labelling. Antibodies to the matrix protein clearly labelled assembled virus structures at the plasma membrane but exhibited no significant cytoplasmic or intranuclear reaction. The data indicate that the composition of nucleocapsids varies with the cellular compartment with which they are associated, supporting the view of a rapid assembly of paramyxovirus nucleocapsid polypeptides, and emphasize the proposed selective role of the matrix protein in virus assembly and budding at the plasma membrane.

We report Mössbauer and EPR measurements on horseradish peroxidase in the native state and the reaction intermediates with peroxide and chlorite. A detailed analysis of the electronic state of the heme iron is given, and comparisons are drawn with related systems. The native enzyme is high-spin ferric and thus has three Kramers doublets. The unusual magnetic properties of the ground doublet and the large energy of the second, (E2-E1)/k approximately equal to 41 K, and third doublet, (E3-E1)/k greater than or equal to 170 K, can be modeled with a quartet admixture of approximately 11% to the spin sextet. All evidence suggests a ferryl, OFeIV, state of the heme iron in compounds I and II and related complexes. The small isomer shift, delta Fe approximately equal to 0.06 mm/s, the (positive) quadrupole splitting, delta EQ approximately equal to 1.4 mm/s, the spin S = 1, and the large positive zero field splitting, D/k approximately equal to 35 K, are all characteristic of the ferryl state. In the green compound I the iron weakly couples to a porphyrin radical with spin S' = 1/2. A phenomenological model with a weak exchange interaction S . J . S', magnitude of less than or equal to 0.1 D, reproduces all Mössbauer and EPR data of compound I, but the structural origin of the exchange and its apparent distribution require further study. Reaction of horseradish peroxidase with chlorite leads to compound X with delta Fe = 0.07 mm/s and delta EQ = 1.53 mm/s, values that are closest to those of compound II. The diamagnetism of compound III and its Mössbauer parameters delta Fe = 0.23 mm/s and delta EQ = -2.31 mm/s at 4.2 K clearly identify it as an oxyheme adduct.

The green primary compound of chloroperoxidase was prepared by freeze-quenching the enzyme after rapid mixing with a 5-fold excess of peracetic acid. The electron paramagnetic resonance (EPR) spectra of these preparations consisted of at least three distinct signals that could be assigned to native enzyme, a free radical, and the green compound I as reported earlier. The absorption spectrum of compound I was obtained through subtraction of EPR signals measured under passage conditions. The signal is well approximated by an effective spin Seff = 1/2 model with g = 1.64, 1.73, 2.00 and a highly anisotropic line width. Mössbauer difference spectra of compound I samples minus native enzyme showed well-resolved magnetic splitting at 4.2 K, an isomer shift delta Fe = 0.15 mm/s, and quadrupole splitting delta EQ = 1.02 mm/s. All data are consistent with the model of an exchange-coupled spin S = 1 ferryl iron and a spin S' = 1/2 porphyrin radical. As a result of the large zero field splitting, D, of the ferryl iron and of intermediate antiferromagnetic exchange, S.J.S'.J approximately 1.02 D, the system consists of three Kramers doublets that are widely separated in energy. The model relates the EPR and Mössbauer spectra of the ground doublet to the intrinsic parameters of the ferryl iron, D/k = 52 K, E/D congruent to 0.035, and A perpendicular (gn beta n) = 20 T, and the porphyrin radical.(ABSTRACT TRUNCATED AT 250 WORDS)

From the temperature dependence of the Orbach relaxation rate of the paramagnetic center in horseradish peroxidase (HRP), we deduce an excited-state energy of 40.9 +/- 1.1 K. Similar studies on the broad EPR signal of HRP compound I indicate a much weaker Orbach relaxation process involving an excited state at 36.8 +/- 2.5 K. The strength of the Orbach process in HRP-I is weaker than one would normally estimate by 2-4 orders of magnitude. This fact lends support to the model of HRP-I involving a spin 1/2 free radical coupled to a spin 1 Fe4+ heme iron via a weak exchange interaction. Such a system should exhibit an Orbach relaxation process involving delta E, the excited state of the Fe4+ ion, but reduced in strength by (Jyy/delta E)2, where Jyy is related to the strength of the exchange interaction between the two spin systems.

The electron paramagnetic resonance (EPR) and Mössbauer properties of native horseradish peroxidase have been compared with those of a synthetic derivative of the enzyme in which a mesohemin residue replaces the natural iron protoporphyrin IX heme prosthetic group. The oxyferryl pi cation radical intermediate, compound I, has been formed from both the native and synthetic enzyme, and the magnetic properties of both intermediates have been examined. The optical absorption characteristics of compound I prepared from mesoheme-substituted horseradish peroxidase are different from those of the compound I prepared from native enzyme [DiNello, R. K., & Dolphin, D. (1981) J. Biol. Chem. 256, 6903-6912]. By analogy to model-compound studies, it has been suggested that these optical absorption differences are due to the formation of an A2u and an A1u pi cation radical species, respectively. However, the EPR and Mössbauer properties of the native and synthetic enzyme and of their oxidized intermediates are quite similar, if not identical, and the data favor an A2u radical for both compounds I.

The electron paramagnetic resonance spectra of chloroperoxidase Compound I and native enzyme are compared. Upon the formation of Compound I, the g = 2.62, 2.26, and 1.82 signals associated with native enzyme disappear and are replaced by two new EPR signals, a sharp signal at g = 2.008 and a broad signal at g = 1.73. The g = 2.008 signal accounts for only 2% of the theoretical spins while the broad signal at g = 1.73 accounts for 60 to 70% of the theoretical spins in Compound I. The g = 1.73 broad signal is reminiscent of the broad EPR signal associated with horseradish peroxidase Compound I. however, the chloroperoxidase Compound I signal has a significantly different g value. The results suggest that the g = 1.73 signal represents a porphyrin pi cation radical which has a stronger coupling to the heme ferryl iron than is the case with horseradish peroxidase Compound I.

When the only substrate added to a solution of chloroperoxidase is a hydroperoxide, the reactions are: ferric enzyme + ROOH leads to compound I + ROH and compound I + ROOH leads to ferric enzyme + O2 + ROH. When H2O2 is used as substrate, the rate constants for the formation and catalatic decomposition of compound I are 2.4 X 10(6) M--1.S--1 and 3.4 X 10(5) M--1.S--1 at pH 4.7 and it is predicted that a maximum of 87% of the enzyme converts to compound I in the steady state of the catalatic reaction. With methyl hydroperoxide, formation of compound I has a rate constant of 4.7 X 10(5) M--1.S--1 and its decomposition 2.9 X 10(4) M--1.S--1. When peracetic acid is used, compound I is formed with a rate constant of 3.8 X 10(6) M--1.S--1 and a 100% yield of compound I is obtained.

Electron-nuclear double resonance spectra of horseradish peroxidase Compound I exhibit resonances from 14N and beta-protons, and most probably alpha-protons as well. The measurements prove that the oxyferryl iron of this enzymic intermediate is spin-coupled to a porphyrin radical. Comparison of experimentally obtained average pi-electron spin densities with theoretical predictions for a 4-fold symmetric porphyrin pi-cation radical is supportive of a 2A2u assignment for the radical's electronic state.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTOxygen-17 ENDOR of horseradish peroxidase compound IJames E. Roberts, Brian M. Hoffman, Rick Rutter, and Lowell P. HagerCite this: J. Am. Chem. Soc. 1981, 103, 25, 7654–7656Publication Date (Print):December 1, 1981Publication History Published online1 May 2002Published inissue 1 December 1981https://pubs.acs.org/doi/10.1021/ja00415a044https://doi.org/10.1021/ja00415a044research-articleACS PublicationsRequest reuse permissionsArticle Views270Altmetric-Citations88LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts

When compound I of chloroperoxidase is formed from the native enzyme the absorption peak in the Soret region diminishes in intensity, and shifts to a maximum absorbance at 367 nm. This unusual Soret spectrum decreases in intensity in a linear fashion as the wavelength increases. The first visible spectrum of chloroperoxidase compound I is reported which has a peak at 689 nm as its most prominent feature.

FEBS LettersVolume 103, Issue 1 p. 102-105 Full-length articleFree Access Horseradish peroxidase compound I: evidence for spin coupling between the heme iron and a 'free' radical C.E. Schulz, C.E. Schulz Departments of Physics and Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USASearch for more papers by this authorP.W. Devaney, P.W. Devaney Departments of Physics and Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USASearch for more papers by this authorH. Winkler, H. Winkler Departments of Physics and Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA Present address: II. Institut für Experimentalphysik, Universität Hamburg, 2000 Hamburg 50, FRG Search for more papers by this authorP.G. Debrunner, P.G. Debrunner Departments of Physics and Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USASearch for more papers by this authorN. Doan, N. Doan Departments of Physics and Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USASearch for more papers by this authorR. Chiang, R. Chiang Departments of Physics and Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA Present address: Grace Chemical Company, Baltimore, MD, USA Search for more papers by this authorR. Rutter, R. Rutter Departments of Physics and Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USASearch for more papers by this authorL.P. Hager, L.P. Hager Departments of Physics and Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USASearch for more papers by this author C.E. Schulz, C.E. Schulz Departments of Physics and Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USASearch for more papers by this authorP.W. Devaney, P.W. Devaney Departments of Physics and Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USASearch for more papers by this authorH. Winkler, H. Winkler Departments of Physics and Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA Present address: II. Institut für Experimentalphysik, Universität Hamburg, 2000 Hamburg 50, FRG Search for more papers by this authorP.G. Debrunner, P.G. Debrunner Departments of Physics and Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USASearch for more papers by this authorN. Doan, N. Doan Departments of Physics and Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USASearch for more papers by this authorR. Chiang, R. Chiang Departments of Physics and Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA Present address: Grace Chemical Company, Baltimore, MD, USA Search for more papers by this authorR. Rutter, R. Rutter Departments of Physics and Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USASearch for more papers by this authorL.P. Hager, L.P. Hager Departments of Physics and Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USASearch for more papers by this author First published: July 01, 1979 https://doi.org/10.1016/0014-5793(79)81259-4Citations: 195 AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume103, Issue1July 01, 1979Pages 102-105 ReferencesRelatedInformation