Hazes, Bart

English

Hemocyanin is the oxygen transport pigment in many arthropods and molluscs. In this respect, hemocyanin has the same function as hemoglobin has in many other organisms. This functional homology has resulted in the development of a number of special properties in both protein classes, e.g. cooperative oxygen binding, regulation by several allosteric effectors and the aggegation into muli-meric complexes. However, the molecular basis by which hemocyanins and hemoglobins achieve these properties is completely different. ... Zie: Summar

Proceedings - University of Groningen

   University of GroningenX-ray crystallographic studies on structure and fucntion of hemocyanins.Hazes, BartIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1993Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Hazes, B. (1993). X-ray crystallographic studies on structure and fucntion of hemocyanins. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 03-06-2022SummaryHemocyanin is the oxygen transport pigment in many arthropods and molluscs. In thisrespect, hemocyanin has the same function as hemoglobin has in many other organisms.This functional homology has resulted in the development of a number of specialproperties in both protein classes, e.g. cooperative oxygen binding, regulation byseveral allosteric effectors and the aggegation into muli-meric complexes. However,the molecular basis by which hemocyanins and hemoglobins achieve these properties iscompletely different.In hemocyanin, molecular oxygen is bound to a dinuclear copper site. The copper ionsare directly coordinated by the protein through 6 histidine side-chain atoms. Uponoxygenation, both copper ions are oxidised from Cu(I) to Cu(II). Accordingly, theoxygen binds as a peroxide (O22-;. In the oxygenated state, hemocyanins displayseveral characteristic spectroscopic features, e.g. strong absorption at 340 and 580 nm(the 580 band gives the protein its characteristic blue colour), an unusual low resonanceRaman frequently for the peroxide bond and the absence of the usual Cu(II) EPR-signal.The cooperative oxygen-binding in hemocyanins indicates that the multi-mericprotein complex exists in (at least) two distinct conformations that differ in oxygenaffinity. The equilibrium between these conformations depends on the ligational state ofthe subunits and on the presence of heterotropic allosteric effectors. The linkagebetween changes in the protein structure and changes in the dinuclear copper site formsthe core of this thesis.In chapter 2, the crystal structure of deoxygenated Limulus polyphemus subunit type II(LImulus II) is presented. The oxygenated structure of this protein was determined inthe group of Dr. Magnus (Cleveland, Ohio). A third structure, that of deoxygenatedPanulirus interruptus hemocyanin, had previously been solved in our laboratory.Comparison of the two Limuhrs II structures has allowed to determine the oxygenationlinked changes in the dinuclear copper site. The most striking observation in thisrespect is that, upon oxygenation, the Cu-Cu distance is reduced from 4.6 to 3.6 A. fnecomparison between the Limulus II sffuctures and the Panulirus hemocyanin structurerevealed considerable changes in the tertiary and quaternary structures. On the basis ofthese observations, a regulation mechanism for arthropodan hemocyanins has beenproposed. In this model it is postulated that the Panulirus and Limulus structuresrepresent the high and low affinity conformations respectively. The main differencebetween these two conformations is a7.5" rotation of the amino terminal domain withrespect to the remainder of the protein. This rotation is structurally coupled to thedinuclear copper site by the interaction between Phenylalanine 49 rn domain 1, the'allosteric sensor', and the copper ligand Histidine 328 in domain 2. The rotation ofdomain 1 also gives an elegant explanation for the observed regulation by chloride ions.In chapter 4, it is shown that nitrate can bind at the same position as chloride ions.Considering the structural homology between nitrate and HCO3-, it may be expectedthat HCO3- can also bind at this position. In hemoglobins, HCO3- is known to be animportant allosteric effector. Oxygen-binding studies have to confirm whether or not asimilar regulatory mechanism also exists rn Limulus II hemocyanin.Chapter 4 also proposes an extension to the two-state concerted model for allostericregulation. In this model, two tertiary conformations are allowed within the low affinity-133-quaternary conformation of the hemocyanin hexamer. The new model can explain theobserved regulation properties of chloride ions and protons, which deviate from a two-state concerted model. In addition, the model explains the observed relation betweenregulation by chloride ions and protons.In chapter 5, the initial crystallisation and X-ray diffraction results with the hemocyaninof. Helix pomatia, a mollusc, are described. Electron microscopy and amino acidsequence determinations have shown that the molluscan and arthropodan hemocyaninshave very different structures. The hemocyanin in Helix pomatia is a 20-meric complexwith a molecular mass of -9 MDa. This makes it one of the largest protein complexesthat have been investigated by X-ray crystallography. As a consequence, the datacollection and processing turned out to be non-trivial tasks. The problems encounteredand the first results will be presented.As a side-study, the folding of Greek key B-banel proteins was investigated based onthe structural homology between the Greek key p-banels observed in: the C-terminaldomain of arthropodan hemocyanins, immuno globulins, Cu,Zn-superoxide dismutase,and a class of anti-tumour proteins. These 4 protein classes do not have a knownevolutionary of functional relationship. Accordingly, it was assumed that conservedfeatures in their amino acid sequences would be related to their structural homology.Analysis of the amino acid sequences, after alignment based on the structuralhomology, showed that the sequences of these proteins are very dissimilar. However,we observed to conserved patterns of large hydrophobic residues in alternatingpositions, separated by a short peptide. This patterns represent he two central stands inthe b-barrel. The conservation of side-chain size and loop length are remarkable, sinceother studies have shown that these features are normally not well conserved. Theconservation of these features can also not be explained satisfactorily fromthermodynamic considerations. In contrast, the conserved side-chain size and looplength fits well within a model where the two central b-strands play an important role inthe nucleation of protein folding.-134-

X-ray crystallographic studies on structure and fucntion of hemocyanins.

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X-ray crystallographic studies on structure and fucntion of hemocyanins.

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