Enzyme Mechanisms: Fast Reaction and Computational Approaches Ultrafast catalytic processes and conformational changes in the light-driven enzyme protochlorophyllide oxidoreductase (POR)

Abstract The enzyme POR (protochlorophyllide oxidoreductase), from the family of alcohol dehydrogenases, reduces protochlorophyllide into chlorophyllide on the absorption of light. The reduction involves the transfer of two protons and two electrons and is an important regulatory step in the biosynthesis of chlorophyll. In recent years, due to the availability of large quantities of the pure enzyme, much of the catalytic reaction has been unravelled by using a variety of spectroscopic methods, including ultrafast initial events in catalysis. In addition, it has been demonstrated that a light-activated conformational change of the protein is necessary to activate catalysis. This makes POR a very important model system to study the relationship between structural changes of enzymes and functionality

Interplay between chromophore binding and domain assembly by the B₁₂-dependent photoreceptor protein, CarH.

From Europe PMC via Jisc Publications RouterHistory: ppub 2021-05-01, epub 2021-05-05Publication status: PublishedFunder: Biotechnology and Biological Sciences Research Council; Grant(s): BB/L002655/1, BB/L016486/1, BB/M011208/1Organisms across the natural world respond to their environment through the action of photoreceptor proteins. The vitamin B12-dependent photoreceptor, CarH, is a bacterial transcriptional regulator that controls the biosynthesis of carotenoids to protect against photo-oxidative stress. The binding of B12 to CarH monomers in the dark results in the formation of a homo-tetramer that complexes with DNA; B12 photochemistry results in tetramer dissociation, releasing DNA for transcription. Although the details of the response of CarH to light are beginning to emerge, the biophysical mechanism of B12-binding in the dark and how this drives domain assembly is poorly understood. Here - using a combination of molecular dynamics simulations, native ion mobility mass spectrometry and time-resolved spectroscopy - we reveal a complex picture that varies depending on the availability of B12. When B12 is in excess, its binding drives structural changes in CarH monomers that result in the formation of head-to-tail dimers. The structural changes that accompany these steps mean that they are rate-limiting. The dimers then rapidly combine to form tetramers. Strikingly, when B12 is scarcer, as is likely in nature, tetramers with native-like structures can form without a B12 complement to each monomer, with only one apparently required per head-to-tail dimer. We thus show how a bulky chromophore such as B12 shapes protein/protein interactions and in turn function, and how a protein can adapt to a sub-optimal availability of resources. This nuanced picture should help guide the engineering of B12-dependent photoreceptors as light-activated tools for biomedical applications

Design and fabrication of recombinant reflectin-based multilayer reflectors: bio-design engineering and photoisomerism induced wavelength modulation

From Springer Nature via Jisc Publications RouterHistory: received 2020-10-03, accepted 2021-06-18, registration 2021-07-07, pub-electronic 2021-07-16, online 2021-07-16, collection 2021-12Publication status: PublishedFunder: Defence Science and Technology Laboratory; doi: http://dx.doi.org/10.13039/100010418Funder: Engineering and Physical Sciences Research Council; doi: http://dx.doi.org/10.13039/501100000266; Grant(s): EP/S01778X/1, EP/S01778X/1, EP/S01778X/1Abstract: The remarkable camouflage capabilities of cephalopods have inspired many to develop dynamic optical materials which exploit certain design principles and/or material properties from cephalopod dermal cells. Here, the angle-dependent optical properties of various single-layer reflectin thin-films on Si wafers are characterized within the UV–Vis–NIR regions. Following this, initial efforts to design, fabricate, and optically characterize a bio-inspired reflectin-based multilayer reflector is described, which was found to conserve the optical properties of single layer films but exhibit reduced angle-dependent visible reflectivity. Finally, we report the integration of phytochrome visible light-induced isomerism into reflectin-based films, which was found to subtly modulate reflectin thin-film reflectivity

The first catalytic step of the light-driven enzyme protochlorophyllide oxidoreductase proceeds via a charge transfer complex

In chlorophyll biosynthesis protochlorophyllide reductase (POR) catalyzes the light-driven reduction of protochlorophyllide (Pchlide) to chlorophyllide, providing a rare opportunity to trap and characterize catalytic intermediates at low temperatures. Moreover, the presence of a chlorophyll-like molecule allows the use of EPR, electron nuclear double resonance, and Stark spectroscopies, previously used for the analysis of photosynthetic systems, to follow catalytic events in the active site of POR. Different models involving the formation of either radical species or charge transfer complexes have been proposed for the initial photochemical step, which forms a nonfluorescent intermediate absorbing at 696 nm (

Single crystal spectroscopy and multiple structures from one crystal (MSOX) define catalysis in copper nitrite reductases

Many enzymes utilize redox-coupled centers for performing catalysis where these centers are used to control and regulate the transfer of electrons required for catalysis, whose untimely delivery can lead to a state incapable of binding the substrate, i.e., a dead-end enzyme. Copper nitrite reductases (CuNiRs), which catalyze the reduction of nitrite to nitric oxide (NO), have proven to be a good model system for studying these complex processes including proton-coupled electron transfer (ET) and their orchestration for substrate binding/utilization. Recently, a two-domain CuNiR from a Rhizobia species (Br2DNiR) has been discovered with a substantially lower enzymatic activity where the catalytic type-2 Cu (T2Cu) site is occupied by two water molecules requiring their displacement for the substrate nitrite to bind. Single crystal spectroscopy combined with MSOX (multiple structures from one crystal) for both the as-isolated and nitrite-soaked crystals clearly demonstrate that inter-Cu ET within the coupled T1Cu-T2Cu redox system is heavily gated. Laser-flash photolysis and optical spectroscopy showed rapid ET from photoexcited NADH to the T1Cu center but little or no inter-Cu ET in the absence of nitrite. Furthermore, incomplete reoxidation of the T1Cu site (∼20% electrons transferred) was observed in the presence of nitrite, consistent with a slow formation of NO species in the serial structures of the MSOX movie obtained from the nitrite-soaked crystal, which is likely to be responsible for the lower activity of this CuNiR. Our approach is of direct relevance for studying redox reactions in a wide range of biological systems including metalloproteins that make up at least 30% of all proteins

Human Cryptochrome-1 Confers Light Independent Biological Activity in Transgenic Drosophila Correlated with Flavin Radical Stability

Cryptochromes are conserved flavoprotein receptors found throughout the biological kingdom with diversified roles in plant development and entrainment of the circadian clock in animals. Light perception is proposed to occur through flavin radical formation that correlates with biological activity in vivo in both plants and Drosophila. By contrast, mammalian (Type II) cryptochromes regulate the circadian clock independently of light, raising the fundamental question of whether mammalian cryptochromes have evolved entirely distinct signaling mechanisms. Here we show by developmental and transcriptome analysis that Homo sapiens cryptochrome - 1 (HsCRY1) confers biological activity in transgenic expressing Drosophila in darkness, that can in some cases be further stimulated by light. In contrast to all other cryptochromes, purified recombinant HsCRY1 protein was stably isolated in the anionic radical flavin state, containing only a small proportion of oxidized flavin which could be reduced by illumination. We conclude that animal Type I and Type II cryptochromes may both have signaling mechanisms involving formation of a flavin radical signaling state, and that light independent activity of Type II cryptochromes is a consequence of dark accumulation of this redox form in vivo rather than of a fundamental difference in signaling mechanism

FACTORS AFFECTING MIGRATION FROM THE CROATIAN RURAL AREA

U radu se daju rezultati istraživanja migracija u seoskom području Republike Hrvatske. Cilj je istražiti čimbenike koji utječu na iseljavanje seoskog stanovništva Republike Hrvatske. Istraživanje je provedeno 2007. godine na uzorku od 914 ispitanika dobi od 24 do 45 godina u seoskom području Republike Hrvatske. Odabir naselja i ispitanika bio je slučajan. Provedeno istraživanje pokazuje da su najveće poteškoće života u hrvatskom seoskom području gospodarske naravi, manjak zaposlenja, slaba mogućnost izbora zanimanja i niža zarada u odnosu na zaposlenje u gradu. Petina ispitanika nije zadovoljna uvjetima seoskog života i namjerava se iseliti. To je zabrinjavajući pokazatelj budući da se radi o populaciji koja je u pravilu završila proces obrazovanja i većinom osnovala obitelj. Najviše mogućih iseljenika, što je bilo i za očekivati, je iz gospodarski nerazvijenih područja Republike Hrvatske. Daljnja depopulacija hrvatskog sela bila bi pogubna, a njene najveće posljedice bile bi: prevelika urbanizacija, posebice velikih gradova, daljnji neravnomjerni razvitak Republike Hrvatske te nedovoljno iskorištenje prostornog, proizvodnog i ljudskog potencijala. S obzirom na strateški cilj ulaska Republike Hrvatske u Europsku uniju, navedeno predstavlja bitno ograničenje njene uspješne prilagodbe europskoj ekonomskoj integraciji. Iseljavanje seoskog pučanstva može se spriječiti prvenstveno povećanjem zaposlenosti i dohotka te stvaranjem takve fizičke i društvene infrastrukture u seoskom području koja će bitno poboljšati životne uvjete seoskog pučanstva. Seoska područja, poglavito gospodarski nerazvijena, nemaju dovoljno vlastitih mogućnosti za ubrzanje razvoja odnosno za nužno smanjivanje razlika u kakvoći življenja prema gradskim područjima. Zbog toga je nužno da njihov razvojni proces više nego dosada potpomogne Država osmišljenim mjerama regionalnog razvoja, uz svekoliku potporu lokalne uprave i samouprave. U tome bi svoj znatan obol trebalo dati novo-osnovano Ministarstvo za regionalni razvoj.The paper presents results of the research study on migrations in rural areas of the Republic of Croatia. The aim was to determine factors influencing migrations of rural population in Croatia. The research was carried out in 2007 on 914 respondents from 25 to 45 years of age. The rural communities and respondents were selected on a random basis. The study results indicate that the major difficulties in rural life in Croatia are of economic nature: lack of employment opportunities, inadequate choice of profession and lower income in comparison with employment in urban areas. One fifth of the respondents is not satisfied with conditions of rural life and intends to leave villages. This is a very disturbing indicator, since it refers to population, which in general, has finished education and started a family. As we expect, the largest number of potential migrants comes from economically underdeveloped Croatian areas. Further depopulation of Croatian villages would have dramatic effects, and the worst consequences would be excessive urbanization, especially of large cities, further uneven development of the Republic of Croatia, and insufficient utilization of spatial, production and human resources. Since the strategic Croatian goal is to become a member of the European Union, this is a major obstacle to its successful adjustment to the European economic integration. The migration of rural population could be prevented primarily by increase in employment and income opportunities and creation of such physical and social infrastructure in rural areas that would considerably improve living conditions for rural population. The rural areas, particularly underdeveloped, have no adequate capacities for intensification of its development and diminishing differences in their quality of life compared to urban areas. Thus, the state support is increasingly required by introducing measures of regional development with complementary support of the local government. The newly founded Ministry of Regional Development is therefore inevitable in this process