Academic literature on the topic 'Heme-binding protein 2'

Heme oxygenase-2 (HO2) and -1 (HO1) catalyze heme degradation to biliverdin, CO, and iron, forming an essential link in the heme metabolism network. Tight regulation of the cellular levels and catalytic activities of HO1 and HO2 is important for maintaining heme homeostasis. HO1 expression is transcriptionally regulated; however, HO2 expression is constitutive. How the cellular levels and activity of HO2 are regulated remains unclear. Here, we elucidate the mechanism of post-translational regulation of cellular HO2 levels by heme. We find that, under heme-deficient conditions, HO2 is destabilized and targeted for degradation, suggesting that heme plays a direct role in HO2 regulation. HO2 has three heme binding sites: one at its catalytic site and the others at its two heme regulatory motifs (HRMs). We report that, in contrast to other HRM-containing proteins, the cellular protein level and degradation rate of HO2 are independent of heme binding to the HRMs. Rather, under heme deficiency, loss of heme binding to the catalytic site destabilizes HO2. Consistently, an HO2 catalytic site variant that is unable to bind heme exhibits a constant low protein level and an enhanced protein degradation rate compared with the WT HO2. Finally, HO2 is degraded by the lysosome through chaperone-mediated autophagy, distinct from other HRM-containing proteins and HO1, which are degraded by the proteasome. These results reveal a novel aspect of HO2 regulation and deepen our understanding of HO2's role in maintaining heme homeostasis, paving the way for future investigation into HO2's pathophysiological role in heme deficiency response.

Iron is one of the essential elements for all living organisms. Pathogenic bacteria acquire heme from the host proteins as an iron source. Gram-negative opportunistic pathogen, Burkholderia cenocepacia have ATP-binding cassette (ABC) transporter BhuUV-T complex to permeate heme through inner membrane. BhuT, periplasmic binding protein (PBP), bind and deliver heme(s) to inner membrane transporter BhuUV complex. BhuUV is 2:2 complex of the transmembrane permease subunit and cytoplasmic ATP-binding subunit which couple ATP hydrolysis to solute translocation. The molecular level mechanism of heme recognition and dissociation by PBP and heme transport by transporter complex are not fully understood. Here we describe the crystal structures of the heme-free and two types of heme-bound state of BhuT. These crystals were obtained in different crystallization conditions. Crystals diffracted to high resolution at SPring-8. BhuT is composed of two globular domains linked by a long a-helix. The transport ligand heme is bound between the two domains. A detailed structural comparison of the conformation of the domain and residues involved in the heme binding will be presented.

In the murine venous thrombosis model induced by ligation of the inferior vena cava (IVCL), genetic deficiency of heme oxygenase-1 (HO-1) increases clot size. This study examined whether induction of HO-1 or administration of its products reduces thrombosis. Venous HO-1 upregulation by gene delivery reduced clot size, as did products of HO activity, biliverdin, and carbon monoxide. Induction of HO-1 by hemin reduced clot formation, clot size, and upregulation of plasminogen activator inhibitor-1 (PAI-1) that occurs in the IVCL model, while leaving urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA) expression unaltered. The reductive effect of hemin on clot size required HO activity. The IVCL model exhibited relatively high concentrations of heme that peaked just before maximum clot size, then declined as clot size decreased. Administration of hemin decreased heme concentration in the IVCL model. HO-2 mRNA was induced twofold in the IVCL model (vs. 40-fold HO-1 induction), but clot size was not increased in HO-2−/− mice compared with HO-2+/+ mice. Hemopexin, the major heme-binding protein, was induced in the IVCL model, and clot size was increased in hemopexin−/− mice compared with hemopexin+/+ mice. We conclude that in the IVCL model, the heme-degrading protein HO-1 and HO products inhibit thrombus formation, as does the heme-binding protein, hemopexin. The reductive effects of hemin administration require HO activity and are mediated, in part, by reducing PAI-1 upregulation in the IVCL model. We speculate that HO-1, HO, and hemopexin reduce clot size by restraining the increase in clot concentration of heme (now recognized as a procoagulant) that otherwise occurs. NEW & NOTEWORTHY This study provides conclusive evidence that two proteins, one heme-degrading and the other heme-binding, inhibit clot formation. This may serve as a new therapeutic strategy in preventing and treating venous thromboembolic disease.

A variety of heme-containing gas sensor proteins have been discovered by gene analysis from bacteria to mammals. In general, these proteins are composed of an N-terminal heme-containing sensor domain and a C-terminal catalytic domain. Binding of O2, CO, or NO to the heme causes a change in the structure of heme, which alters the protein conformation in the vicinity of the heme, and the conformational change is propagated to the catalytic domain, leading to regulation of the protein activity. This mini-review summarizes the recent resonance Raman studies obtained with both visible and UV excitation sources for two O2 sensor proteins, EcDOS and HemAT-Bs. These investigations have shown the role of heme propionate hydrogen-bonding interactions in communicating the heme structural changes, which occur upon ligand binding, from heme to the protein moiety. Furthermore, it is deduced that the contact interactions between the heme 2-vinyl group and the surrounding residues are also important for signal transmission from heme to protein in EcDOS.

Absorption and magnetic circular dichroism (MCD) spectra, together with electrospray ionization mass spectral (ESI-MS) data are reported for the first two proteins in the Isd sequence of proteins in Staphylococcus aureus. IsdH-NEAT domain 3 (IsdH-N3) and IsdB-NEAT domain 2 (IsdB-N2) are considered to be involved in heme transport following heme scavenging from the hemoglobin of the host. The ESI-MS data show that a single heme binds to each of these NEAT domains. The charge states of the native proteins indicate that there is minimal change in conformation when heme binds to the heme-free native protein. Acid denaturation releases the bound heme and results in protein that exhibits significantly higher charge states, which we associate with unfolding of the protein structure. MCD spectra of the heme-bound native proteins show that the heme-iron is in a high-spin state, which is similar to that in IsdC-N. Addition of cyanide results in a spectral envelope characteristic of low-spin ferric hemes. The lack of complete binding for IsdH-N3 suggests that there is considerable congestion in the heme-binding site region. Unusually, reduction to the ferrous heme results in spectral characteristics of six coordination of the ferrous heme. CO is shown to bind strongly to both heme bound proteins, resulting in six-coordinate bound hemes. The spectra following reduction most closely resemble spectra recorded for heme with histidine in the fifth position and methionine in the sixth position. We report a theoretical model calculated from the X-ray structure coordinates of IsdH-N3, in which the heme is coordinated to nearby histidine and methionine. We propose that this structure accounts for the spectroscopic properties of the protein with the ferrous heme.

The circadian clock is an endogenous time-keeping system that is ubiquitous in animals and plants as well as some bacteria. In mammals, the clock regulates the sleep–wake cycle via 2 basic helix–loop–helix PER-ARNT-SIM (bHLH-PAS) domain proteins—CLOCK and BMAL1. There is emerging evidence to suggest that heme affects circadian control, through binding of heme to various circadian proteins, but the mechanisms of regulation are largely unknown. In this work we examine the interaction of heme with human CLOCK (hCLOCK). We present a crystal structure for the PAS-A domain of hCLOCK, and we examine heme binding to the PAS-A and PAS-B domains. UV-visible and electron paramagnetic resonance spectroscopies are consistent with a bis-histidine ligated heme species in solution in the oxidized (ferric) PAS-A protein, and by mutagenesis we identify His144 as a ligand to the heme. There is evidence for flexibility in the heme pocket, which may give rise to an additional Cys axial ligand at 20K (His/Cys coordination). Using DNA binding assays, we demonstrate that heme disrupts binding of CLOCK to its E-box DNA target. Evidence is presented for a conformationally mobile protein framework, which is linked to changes in heme ligation and which has the capacity to affect binding to the E-box. Within the hCLOCK structural framework, this would provide a mechanism for heme-dependent transcriptional regulation.

Heme-regulatory motifs (HRMs) are present in many proteins that are involved in diverse biological functions. The C-terminal tail region of human heme oxygenase-2 (HO2) contains two HRMs whose cysteine residues form a disulfide bond; when reduced, these cysteines are available to bind Fe3+-heme. Heme binding to the HRMs occurs independently of the HO2 catalytic active site in the core of the protein, where heme binds with high affinity and is degraded to biliverdin. Here, we describe the reversible, protein-mediated transfer of heme between the HRMs and the HO2 core. Using hydrogen-deuterium exchange (HDX)-MS to monitor the dynamics of HO2 with and without Fe3+-heme bound to the HRMs and to the core, we detected conformational changes in the catalytic core only in one state of the catalytic cycle—when Fe3+-heme is bound to the HRMs and the core is in the apo state. These conformational changes were consistent with transfer of heme between binding sites. Indeed, we observed that HRM-bound Fe3+-heme is transferred to the apo-core either upon independent expression of the core and of a construct spanning the HRM-containing tail or after a single turnover of heme at the core. Moreover, we observed transfer of heme from the core to the HRMs and equilibration of heme between the core and HRMs. We therefore propose an Fe3+-heme transfer model in which HRM-bound heme is readily transferred to the catalytic site for degradation to facilitate turnover but can also equilibrate between the sites to maintain heme homeostasis.

(1) Background: coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been linked to hematological dysfunctions, but there are little experimental data that explain this. Spike (S) and Nucleoprotein (N) proteins have been putatively associated with these dysfunctions. In this work, we analyzed the recruitment of hemoglobin (Hb) and other metabolites (hemin and protoporphyrin IX-PpIX) by SARS-Cov2 proteins using different approaches. (2) Methods: shotgun proteomics (LC–MS/MS) after affinity column adsorption identified hemin-binding SARS-CoV-2 proteins. The parallel synthesis of the peptides technique was used to study the interaction of the receptor bind domain (RBD) and N-terminal domain (NTD) of the S protein with Hb and in silico analysis to identify the binding motifs of the N protein. The plaque assay was used to investigate the inhibitory effect of Hb and the metabolites hemin and PpIX on virus adsorption and replication in Vero cells. (3) Results: the proteomic analysis by LC–MS/MS identified the S, N, M, Nsp3, and Nsp7 as putative hemin-binding proteins. Six short sequences in the RBD and 11 in the NTD of the spike were identified by microarray of peptides to interact with Hb and tree motifs in the N protein by in silico analysis to bind with heme. An inhibitory effect in vitro of Hb, hemin, and PpIX at different levels was observed. Strikingly, free Hb at 1mM suppressed viral replication (99%), and its interaction with SARS-CoV-2 was localized into the RBD region of the spike protein. (4) Conclusions: in this study, we identified that (at least) five proteins (S, N, M, Nsp3, and Nsp7) of SARS-CoV-2 recruit Hb/metabolites. The motifs of the RDB of SARS-CoV-2 spike, which binds Hb, and the sites of the heme bind-N protein were disclosed. In addition, these compounds and PpIX block the virus’s adsorption and replication. Furthermore, we also identified heme-binding motifs and interaction with hemin in N protein and other structural (S and M) and non-structural (Nsp3 and Nsp7) proteins.

Iron regulatory protein 2 coordinates the cellular regulation of iron metabolism by binding to iron-responsive elements in mRNA. The protein is synthesized constitutively but is rapidly degraded when iron stores are replete. The mechanisms that prevent degradation during iron deficiency or promote degradation during iron sufficiency are not delineated. Iron regulatory protein 2 contains a domain not present in the closely related iron regulatory protein 1, and we found that this domain binds heme with high affinity. A cysteine within the domain is axially liganded to the heme, as occurs in cytochrome P450. The protein-bound heme reacts with molecular oxygen to mediate the oxidation of cysteine, including β-elimination of the sulfur to yield alanine. This covalent modification may thus mark the protein molecule for degradation by the proteasome system, providing another mechanism by which heme can regulate the level of iron regulatory protein 2.

ABSTRACT Although efforts have been made to identify circadian-controlled genes regulating cell cycle progression and cell death, little is known about the metabolic signals modulating circadian regulation of gene expression. We identify heme, an iron-containing prosthetic group, as a regulatory ligand controlling human Period-2 (hPer2) stability. Furthermore, we define a novel heme-regulatory motif within the C terminus of hPer2 (SC841PA) as necessary for heme binding and protein destabilization. Spectroscopy reveals that whereas the PAS domain binds to both the ferric and ferrous forms of heme, SC841PA binds exclusively to ferric heme, thus acting as a redox sensor. Consequently, binding prevents hPer2 from interacting with its stabilizing counterpart cryptochrome. In vivo, hPer2 downregulation is suppressed by inhibitors of heme synthesis or proteasome activity, while SA841PA is sufficient to stabilize hPer2 in transfected cells. Moreover, heme binding to the SC841PA motif directly impacts circadian gene expression, resulting in altered period length. Overall, the data support a model where heme-mediated oxidation triggers hPer2 degradation, thus controlling heterodimerization and ultimately gene transcription.

In questo lavoro di tesi ci si è occupati dell’espressione, della purificazione e della cristallizzazione di tre proteine umane (l’apolipoproteina M, il recettore del folato α e la proteina SOUL) con lo scopo finale di determinarne la struttura tridimensionale mediante analisi di diffrazione di raggi X. L’apolipoproteina M umana è stata espressa utilizzando il lievito metilotrofico P. pastoris. La proteina ricombinante così ottenuta è stata purificata tramite cromatografia a scambio ionico, isoelettrofocalizzazione preparativa, gel filtrazione e cromatografia ad interazione idrofobica. Per ottenere una proteina più omogenea è stato espresso e purificato anche il mutante Asn135Gln, privo del sito di glicosilazione. Le prove di cristallizzazione hanno dato esito positivo con la proteina mutata, anche se i cristalli fino ad ora ottenuti non sono idonei per gli esperimenti di diffrazione di raggi X. L’espressione eterologa del recettore del folato umano ha dato parecchi problemi, nonostante siano stati provati diversi sistemi di espressione (P. pastoris, baculovirus e N. benthamiana). Solo una piccola quantità di proteina ricombinante è stata ottenuta (da P. pastoris) e purificata (mediante cromatografia a scambio ionico e gel filtrazione). Nessuna delle condizioni di cristallizzazione testata ha avuto successo, probabilmente a causa della bassa concentrazione della proteina utilizzata in tali prove. La proteina SOUL (heme-binding protein 2) è stata espressa in E. coli e purificata tramite cromatografia di affinità, sfruttando la coda di sei istidine aggiunta all’estremità C-terminale della proteina. La SOUL ricombinante è stata cristallizzata sia come apopoteina sia come oloproteina (complesso SOUL/emina). Gli studi preliminari di diffrazione di raggi X mostrano la presenza di sei molecole nella cella unitaria. Non è stata inoltre rilevata alcuna significativa differenza tra la forma apo- e la forma olo-. Ulteriori studi suggeriscono che l’emina non sia legata alla proteina, poiché il picco corrispondente al ferro non è stato trovato nello spettro di fluorescenza ai raggi X ottenuto dai cristalli. Al momento sono in corso i tentativi di risolverne la struttura tridimensionale per mezzo di sostituzione isomorfa multipla, multiwavelength anomalous diffraction e sostituzione molecolare.
This thesis work was aimed at the expression, purification and crystallization of three human proteins (apolipoprotein M, folate receptor α and SOUL protein) in order to determine their three-dimensional structure by means of X-ray protein crystallography. Human apolipoprotein M was expressed using the methylotrophic yeast P. pastoris. The recombinant protein was purified by ion-exchange chromatography, preparative isoelectric focusing, gel filtration, and Lipidex-1000 chromatography. In order to obtain a more homogeneous protein, the non-glycosylated mutant (Asn135Gln) was also expressed and purified. The crystallization trials gave some positive results with mutated apoM, although the crystals are still not suitable for X-ray diffraction experiments. The heterologous expression of the human FR-a was troublesome, and although different expression systems (P. pastoris, baculovirus, and N. benthamiana) were tested, only a low amount of recombinant protein was obtained (from P. pastoris) and purified (by ion-exchange chromatography and gel filtration). However non of the crystallization conditions tested was successful, probably due to the low protein concentration. Human SOUL (heme-binding protein 2) was expressed in E. coli and purified by immobilized metal ion affinity chromatography, using the hexa-histidine tag added to the C-terminus of the protein. The recombinant SOUL was crystallized both as apoprotein and as a complex in the presence of hemin. The preliminary X-ray diffraction analysis shows the presence of six molecules in the unit cell, and no significant differences between the apoand the holoprotein were found. Further studies suggest that hemin is not bound to the protein, since the Fe peak could not be found in the X-ray fluorescence spectrum of the crystals. Attempts to solve the three-dimensional structure by means of multiple isomorphous replacement, multiwavelength anomalous diffraction and molecular replacement are still in progress.

The effects of high pressure up to 1500 bar on the recombination kinetics of oxygen and carbon monoxide (CO) binding to human hemoglobin (intact and isolated chain forms), human myoglobin (and its mutants), and cytochrome P-450 were studied by the use of millisecond and nanosecond laser photolysis. The activation volumes for the binding of CO to the R- and T-quaternary states of hemoglobin (Hbs) were determined to be –9.0 and –31.7 ml, respectively. The characteristic pressure dependence of the activation volume was observed for the R-state Hb but not for the T-state Hb. More detailed studies were made with isolated α- and β-chains of human Hb. The kinetic data were analyzed on the basis of a simple three-species model, which assumes two elementary reaction processes of bond formation and steps of ligand migration. A pressure-dependent activation volume change from negative lo positive values in the bimolecular CO association reaction was observed for both chains. This is attributed to a change of the rate-limiting step from the bond-formation step to the ligandmigration step. High-pressure ligand-binding kinetics were also examined for site-specific mutants of human myoglobin in which some amino acid residues at the heme distal sites, such as Leu 29, Lys 45, Ala 66, and Thr 67, are substituted by others. The pressure dependence of the CO binding rate for the L29 mutants was unusual: a positive value was obtained unexpectedly for overall CO binding. Corresponding to this anomaly was an unusual geometry of the iron-bound CO, which was determined by IR and NMR spectroscopies. The effects of camphor and camphor analogues as substrates on the CO-binding kinetics for P-450cam were also studied under pressure. The positive activation volumes for CO binding were obtained for substrate-free and norcamphor- and adamantane-bound P-450, whereas other substrate analogue-bound P-450 complexes exhibited the negative activation volumes. All of the present high-pressure results are discussed in relation to (1) the dynamic aspects of the protein conformation, and (2) the specific participation of amino acid residues in the heme distal site in each elementary step of the ligand-binding reaction process.

A critical feature of the biological function of heme proteins is the direct coupling of protein motion to the process of binding exogenous ligands to the heme. In carbonmonoxymyoglobin (MbCO), a substantial, specific conformational relaxation is associated with the transition from the ligated to the unligated form of the protein. The analogous tertiary structural changes of the monomer heme subunits of hemoglobin ultimately lead to the R→T quaternary structural transition, the allosteric control mechanism of O2 binding efficiency [1]. We have studied these processes on the earliest timescales, using picosecond, time-resolved infrared (TRIR) spectroscopy. It has long been known that infrared spectra in the amide region are sensitive to protein secondary conformation [2]. Recent advances in equipment and techniques have permitted researchers to quantitatively predict secondary structures from infrared spectra [3,4], particularly in the amide I region [4]. Therefore, it is now possible to study protein motion in time-resolved experiments on dynamics and function. The ligation reactions of small molecules such as CO with the heme site of Mb exemplify the mechanisms available to O2. CO is an ideal candidate for initial time-resolved IR experiments in the amide I region because it is easily photolyzed, little geminate recombination [5], and the structure of both MbCO and unligated Mb have been studied by crystallographic methods [6]. TRIR has already been applied to the stretching vibrations of the bound and free CO ligand [7,8]; dynamics of the protein, however, have yet to be probed by TRIR spectroscopy of the protein vibrations. Here we report results on the motions of the protein in response to ligation reactions, probed in the amide I region centered about 1650 cm-1.

A fundamental understanding of condensed phase chemical reaction dynamics can be obtained from the study of biomolecules, particularly heme and heme proteins. The complexity of these systems gives rise to a rich variety of phenomena, allowing many aspects of condensed matter reactions to be examined. The rate theories and puzzles of hemeprotein kinetics have recently been discussed by Frauenfelder and Wolynes [1]. In this work we discuss new experiments on ligand binding to protoheme (Fe:protoporphyrin-IX) in a glassy matrix. We have studied the rebinding of carbon monoxide over a wide range of time [5ps-10ms] and temperature [300K–70K]. The significance of our results are that (1) the influence of friction and nonadibaticity on a condensed phase reaction can be directly investigated, (2) the influence of an inhomogeneous glassy matrix (glycerol-water 75:25) on the reaction can be studied, and (3) meaningful comparison between protoheme and hemeprotein kinetics isolates the role of the protein relaxation in the reaction.

Large amplitude motions in proteins and biopolymers is a fundamental feature of the biological function of these systems. The mechanism of energy transduction from a stimulus, such as ligand binding or dissociation, to a specific protein motion associated with biological function remains a controversial issue. Large amplitude motions often involve the correlated action of a vast number of vibrational modes and energy must be directed along specific channels in order to displace a sizable number of atoms. This motion must involve energy delocalization, either over a specific vibrational coordinate as postulated by the soliton model,1 or as delocalized potential energy gradients, such as in the strain model.2 These two possible mechanisms can be distinguished by the spatial dispersion of vibrational energy and the lifetimes of the vibrational modes that couple to the correlated motion. This problem can be ideally studied using transient grating spectroscopy by taking advantage of the high sensitivity and time resolution of this technique to density changes.

The aim of this research project was to obtain information on the role of the cytochrome b559 in the function of Photosystem-II (PSII) with special emphasis on the light induced photo inactivation of PSII and turnover of the photochemical reaction center II protein subunit RCII-D1. The major goals of this project were: 1) Isolation and sequencing of the Chlamydomonas chloroplast psbE and psbF genes encoding the cytochrome b559 a and b subunits respectively; 2) Generation of site directed mutants and testing the effect of such mutation on the function of PSII under various light conditions; 3) To obtain further information on the mechanism of the light induced degradation and replacement of the PSII core proteins. This information shall serve as a basis for the understanding of the role of the cytochrome b559 in the process of photoinhibition and recovery of photosynthetic activity as well as during low light induced turnover of the D1 protein. Unlike in other organisms in which the psbE and psbF genes encoding the a and b subunits of cytochrome b559, are part of an operon which also includes the psbL and psbJ genes, in Chlamydomonas these genes are transcribed from different regions of the chloroplast chromosome. The charge distribution of the derived amino-acid sequences of psbE and psbF gene products differs from that of the corresponding genes in other organisms as far as the rule of "positive charge in" is concerned relative to the process of the polypeptide insertion in the thylakoid membrane. However, the sum of the charges of both subunits corresponds to the above rule possibly indicating co-insertion of both subunits in the process of cytochrome b559 assembly. A plasmid designed for the introduction of site-specific mutations into the psbF gene of C. reinhardtii. was constructed. The vector consists of a DNA fragment from the chromosome of C. reinhardtii which spans the region of the psbF gene, upstream of which the spectinomycin-resistance-conferring aadA cassette was inserted. This vector was successfully used to transform wild type C. reinhardtii cells. The spectinomycin resistant strain thus obtained can grow autotrophically and does not show significant changes as compared to the wild-type strain in PSII activity. The following mutations have been introduced in the psbF gene: H23M; H23Y; W19L and W19. The replacement of H23 involved in the heme binding to M and Y was meant to permit heme binding but eventually alter some or all of the electron transport properties of the mutated cytochrome. Tryptophane W19, a strictly conserved residue, is proximal to the heme and may interact with the tetrapyrole ring. Therefore its replacement may effect the heme properties. A change to tyrosine may have a lesser affect on the potential or electron transfer rate while a replacement of W19 by leucine is meant to introduce a more prominent disturbance in these parameters. Two of the mutants, FW19L and FH23M have segregated already and are homoplasmic. The rest are still grown under selection conditions until complete segregation will be obtained. All mutants contain assembled and functional PSII exhibiting an increased sensitivity of PSII to the light. Work is still in progress for the detailed characterization of the mutants PSII properties. A tobacco mutant, S6, obtained by Maliga and coworkers harboring the F26S mutation in the b subunit was made available to us and was characterized. Measurements of PSII charge separation and recombination, polypeptide content and electron flow indicates that this mutation indeed results in light sensitivity. Presently further work is in progress in the detailed characterization of the properties of all the above mutants. Information was obtained demonstrating that photoinactivation of PSII in vivo initiates a series of progressive changes in the properties of RCII which result in an irreversible modification of the RCII-D1 protein leading to its degradation and replacement. The cleavage process of the modified RCII-D1 protein is regulated by the occupancy of the QB site of RCII by plastoquinone. Newly synthesized D1 protein is not accumulated in a stable form unless integrated in reassembled RCII. Thus the degradation of the irreversibly modified RCII-D1 protein is essential for the recovery process. The light induced degradation of the RCII-D1 protein is rapid in mutants lacking the pD1 processing protease such as in the LF-1 mutant of the unicellular alga Scenedesmus obliquus. In this case the Mn binding site of PSII is abolished, the water oxidation process is inhibited and harmful cation radicals are formed following light induced electron flow in PSII. In such mutants photo-inactivation of PSII is rapid, it is not protected by ligands binding at the QB site and the degradation of the inactivated RCII-D1 occurs rapidly also in the dark. Furthermore the degraded D1 protein can be replaced in the dark in absence of light driven redox controlled reactions. The replacement of the RCII-D1 protein involves the de novo synthesis of the precursor protein, pD1, and its processing at the C-terminus end by an unknown processing protease. In the frame of this work, a gene previously isolated and sequenced by Dr. Pakrasi's group has been identified as encoding the RCII-pD1 C-terminus processing protease in the cyanobacterium Synechocystis sp. PCC 6803. The deduced sequence of the ctpA protein shows significant similarity to the bovine, human and insect interphotoreceptor retinoid-binding proteins. Results obtained using C. reinhardtii cells exposes to low light or series of single turnover light flashes have been also obtained indicating that the process of RCII-D1 protein turnover under non-photoinactivating conditions (low light) may be related to charge recombination in RCII due to back electron flow from the semiquinone QB- to the oxidised S2,3 states of the Mn cluster involved in the water oxidation process.