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NADH cytochrome b 5 oxidoreductase (Ncb5or) is a cytosolic ferric reductase implicated in diabetes and neurological conditions. Ncb5or comprises cytochrome b 5 (b 5 ) and cytochrome b 5 reductase (b 5 R) domains separated by a CHORD-Sgt1 (CS) linker domain. Ncb5or redox activity depends on proper inter-domain interactions to mediate electron transfer from NADH or NADPH via FAD to heme. While full-length human Ncb5or has proven resistant to crystallization, we have succeeded in obtaining high-resolution atomic structures of the b 5 domain and a construct containing the CS and b 5 R domains (CS/b 5 R). Ncb5or also contains an N-terminal intrinsically disordered region of 50 residues that has no homologs in other protein families in animals but features a distinctive, conserved L 34 MDWIRL 40 motif also present in reduced lateral root formation (RLF) protein in rice and increased recombination center 21 in baker's yeast, all attaching to a b 5 domain. After unsuccessful attempts at crystallizing a human Ncb5or construct comprising the N-terminal region naturally fused to the b 5 domain, we were able to obtain a high-resolution atomic structure of a recombinant rice RLF construct corresponding to residues 25-129 of human Ncb5or (52% sequence identity; 74% similarity). The structure reveals Trp 120 (corresponding to invariant Trp 37 in Ncb5or) to be part of an 11-residue α-helix (S 116 QMDWLKLTRT 126 ) packing against two of the four helices in the b 5 domain that surround heme (α2 and α5). The Trp 120 side chain forms a network of interactions with the side chains of four highly conserved residues corresponding to Tyr 85 and Tyr 88 (α2), Cys 124 (α5), and Leu 47 in Ncb5or. Circular dichroism measurements of human Ncb5or fragments further support a key role of Trp 37 in nucleating the formation of the N-terminal helix, whose location in the N/b 5 module suggests a role in regulating the function of this multi-domain redox enzyme. This study revealed for the first time an ancient origin of a helical motif in the N/b 5 module as reflected by its existence in a class of cytochrome b 5 proteins from three kingdoms among eukaryotes., (© 2023 Wiley Periodicals LLC.)

Cytochrome b 5 reductase 3 (CYB5R3) activates respiratory metabolism in cellular systems and exerts a prolongevity action in transgenic mice overexpressing this enzyme, mimicking some of the beneficial effects of calorie restriction. The aim of our study was to investigate the role of sex on metabolic adaptations elicited by CYB5R3 overexpression, and how key markers related with mitochondrial function are modulated in skeletal muscle, one of the major contributors to resting energy expenditure. Young CYB5R3 transgenic mice did not exhibit the striking adaptations in carbon metabolism previously detected in older animals. CYB5R3 was efficiently overexpressed and targeted to mitochondria in skeletal muscle from transgenic mice regardless sex. Overexpression significantly elevated NADH in both sexes, although differences were not statistically significant for NAD + , and increased the abundance of cytochrome c and the fission protein DRP-1 in females but not in males. Moreover, while mitochondrial biogenesis and function markers (as TFAM, NRF-1 and cleaved SIRT3) were markedly upregulated by CYB5R3 overexpression in females, a downregulation was observed in males. Ultrastructural changes were also highlighted, with an increase in the number of mitochondria per surface unit, and in the size of intermyofibrillar mitochondria in transgenic females compared with their wild-type controls. Our results support that CYB5R3 overexpression upregulates markers consistent with enhanced mitochondrial biogenesis and function, and increases mitochondrial abundance in skeletal muscle, producing most of these potentially beneficial actions in females., Competing Interests: Declaration of competing interest Luz Marina Sánchez-Mendoza, Carlos Pérez-Sánchez, Sandra Rodríguez-López, Chary López-Pedrera, Miguel Calvo-Rubio, Rafael de Cabo, María I. Burón, José A. González-Reyes, and José M. Villalba declare that they have no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Cytochrome b 5 reductase (C b 5 R) is a flavoprotein that participates in the reduction of multiple biological redox partners. Co-localization of this protein with nitric oxide sources has been observed in neurons. In addition, the generation of superoxide anion radical by C b 5 R has been observed. A search for specific inhibitors of C b 5 R to understand the role of this protein in these new functions has been initiated. Previous studies have shown the ability of different flavonoids to inhibit C b 5 R. Anthocyanins are a subgroup of flavonoids responsible for most red and blue colors found in flowers and fruits. Although usually represented by the flavylium cation form, these species are only stable at rather acidic pH values (pH ≤ 1). At higher pH values, the flavylium cation is involved in a dynamic reaction network comprising different neutral species with the potential ability to inhibit the activities of C b 5 R. This study aims to provide insights into the molecular mechanism of interaction between flavonoids and C b 5 R using flavylium salts as dynamic inhibitors. The outcome of this study might lead to the design of improved specific enzyme inhibitors in the future.

Protein modification by ubiquitin-like proteins (UBLs) amplifies limited genome information and regulates diverse cellular processes, including translation, autophagy and antiviral pathways. Ubiquitin-fold modifier 1 (UFM1) is a UBL covalently conjugated with intracellular proteins through ufmylation, a reaction analogous to ubiquitylation. Ufmylation is involved in processes such as endoplasmic reticulum (ER)-associated protein degradation, ribosome-associated protein quality control at the ER and ER-phagy. However, it remains unclear how ufmylation regulates such distinct ER-related functions. Here we identify a UFM1 substrate, NADH-cytochrome b5 reductase 3 (CYB5R3), that localizes on the ER membrane. Ufmylation of CYB5R3 depends on the E3 components UFL1 and UFBP1 on the ER, and converts CYB5R3 into its inactive form. Ufmylated CYB5R3 is recognized by UFBP1 through the UFM1-interacting motif, which plays an important role in the further uyfmylation of CYB5R3. Ufmylated CYB5R3 is degraded in lysosomes, which depends on the autophagy-related protein Atg7- and the autophagy-adaptor protein CDK5RAP3. Mutations of CYB5R3 and genes involved in the UFM1 system cause hereditary developmental disorders, and ufmylation-defective Cyb5r3 knock-in mice exhibit microcephaly. Our results indicate that CYB5R3 ufmylation induces ER-phagy, which is indispensable for brain development., (© 2022. The Author(s).)

The electron-transfer capabilities of cytochrome b 5 reductase (Cyt b 5 R) and NADPH supply have been shown to be critical factors in microbial fatty acid synthesis. Unfortunately, Cyt b 5 R substrate specificity is limited to the coenzyme NADH. In this study, we discovered that a novel Cyt b 5 R from Mortierella alpina ( Ma Cytb5RII) displays affinity for NADPH and NADH. The enzymatic characteristics of high-purity Ma Cytb5RII were determined with the K m, NADPH and K m, NADH being 0.42 and 0.07 mM, respectively. Ma Cytb5RII shows high specific activity at 4 °C and pH 9.0. We anchored the residues that interacted with the coenzymes using the homology models of Ma Cytb5Rs docking NAD(P)H and FAD. The enzyme activity analysis of the purified mutants Ma Cytb5RII [S230N] , Ma Cytb5RII [Y242F] , and Ma Cytb5RII [S272A] revealed that Ser230 is essential for Ma Cytb5RII to have dual NAD(P)H dependence, whereas Tyr242 influences Ma Cytb5RII's NADPH affinity and Ala272 greatly decreases Ma Cytb5RII's NADH affinity.