Bacterial periplasmic nitrate and trimethylamine-N-oxide respiration coupled to menaquinol-cytochrome c reductase (Qcr): Implications for electrogenic reduction of alternative electron acceptors.

The periplasmic reduction of the electron acceptors nitrate (Em +420 mV) and trimethylamine-N-oxide (TMAO; Em +130 mV) by Nap and Tor reductases is widespread in Gram-negative bacteria and is usually considered to be driven by non-energy conserving quinol dehydrogenases. The Epsilonproteobacterium Campylobacter jejuni can grow by nitrate and TMAO respiration and it has previously been assumed that these alternative pathways of electron transport are independent of the proton-motive menaquinol-cytochrome c reductase complex (QcrABC) that functions in oxygen-linked respiration. Here, we show that a qcrABC deletion mutant is completely deficient in oxygen-limited growth on both nitrate and TMAO and is unable to reduce these oxidants with physiological electron donors. As expected, the mutant grows normally on fumarate under oxygen-limited conditions. Thus, the periplasmic Nap and Tor reductases receive their electrons via QcrABC in C. jejuni, explaining the general absence of NapC and TorC quinol dehydrogenases in Epsilonproteobacteria. Moreover, the specific use of menaquinol (Em -75 mV) coupled with a Qcr complex to drive reduction of nitrate or TMAO against the proton-motive force allows the process to be electrogenic with a H+/2e- ratio of 2. The results have general implications for the role of Qcr complexes in bacterial oxygen-independent respiration and growth.

Congenital methemoglobinemia: a rare cause of cyanosis in the newborn--a case report.

Cyanosis is a physical finding that can occur at any age but presents the greatest challenge when it occurs in the newborn. The cause is multiple, and it usually represents an ominous sign, especially when it occurs in association with neonatal sepsis, cyanotic congenital heart disease, and airway abnormalities. Cyanosis caused by abnormal forms of hemoglobin can also be life-threatening, and early recognition is mandatory to prevent unnecessary investigations and delay in management. Abnormal hemoglobin, such as hemoglobin M, is traditionally discovered by electrophoresis, so the newborn screen, which is mandatory in several states, is a useful tool for the diagnosis. Although acquired methemoglobinemia, caused by environmental oxidizing agents, is common, congenital deficiency of the innate reducing enzyme is so rare that only a few cases are documented in the medical literature around the world. We present a neonate with cyanosis as a result of congenital deficiency of the reduced nicotinamide adenine dinucleotide-cytochrome b5 reductase enzyme. This infant was found to be blue at a routine newborn follow-up visit. Sepsis, structural congenital heart disease, prenatal administration, and ingestion of oxidant dyes were excluded as a cause of the cyanosis by history and appropriate tests. Chocolate discoloration of arterial blood provided a clue to the diagnosis. A normal newborn screen and hemoglobin electrophoresis made the diagnosis of hemoglobin M unlikely as the cause of the methemoglobinemia (Hb A 59.4%, A2 1.8%, and F 38.8%). Red blood cell enzyme activity and DNA analysis revealed a homozygous form of the cytochrome b5 reductase enzyme deficiency. He responded very well to daily methylene blue and ascorbic acid administration, and he has normal growth and developmental parameters, although he shows an exaggerated increase in his methemoglobin level with minor oxidant stress such as diarrhea.

Compound heterozygosity of two missense mutations in the NADH-cytochrome b5 reductase gene of a Polish patient with type I recessive congenital methaemoglobinaemia.

A case of type I methaemoglobinaemia observed in a Polish subject with compound heterozygosity for two mutations in the reduced nicotinamide adenine dinucleotide (NADH) cytochrome b5 reductase (b5R) gene is described. One is a novel mutation 647T-->C which leads to substitution of isoleucine by threonine at position 215 (I215T). This maternal mutation was found in several family members. A previously known mutation, 757G-->A, leads to the replacement of valine by methionine at position 252 (V252M). The latter mutation was found also in the father and one of the two brothers. The effects of these mutations were analysed on a model of the human b5R protein obtained by homology modelling. Although both amino acid substitutions are located in the NADH-binding domain, the whole protein structure, especially the region between the flavin adenine dinucleotide and NADH-binding domains, is disturbed. The structural changes in the I215T mutant are less prominent than those in the V252M mutant. We presume that the 647T-->C mutation is a type I mutation, however, it has not been observed in the homozygous state.

Familial idiopathic methemoglobinemia revisited: original cases reveal 2 novel mutations in NADH-cytochrome b5 reductase.

In 1943, the first description of familial idiopathic methemoglobinemia in the United Kingdom was reported in 2 members of one family. Five years later, Quentin Gibson (then of Queen's University, Belfast, Ireland) correctly identified the pathway involved in the reduction of methemoglobin in the family, thereby describing the first hereditary trait involving a specific enzyme deficiency. Recessive congenital methemoglobinemia (RCM) is caused by a deficiency of reduced nicotinamide adenine dinucleotide (NADH)-cytochrome b5 reductase. One of the original propositi with the type 1 disorder has now been traced. He was found to be a compound heterozygote harboring 2 previously undescribed mutations in exon 9, a point mutation Gly873Ala predicting a Gly291Asp substitution, and a 3-bp in-frame deletion of codon 255 (GAG), predicting loss of glutamic acid. A brother and a surviving sister are heterozygous; each bears one of the mutations. Thirty-three different mutations have now been recorded for RCM. The original authors' optimism that RCM would provide material for future genetic studies has been amply justified.

A case of methemoglobinemia type II due to NADH-cytochrome b5 reductase deficiency: determination of the molecular basis.

Clinical, biochemical and molecular findings in a patient with methemoglobinemia type II are described. Furthermore, a comparison between methemoglobinemia type I and type II, both caused by a deficiency of NADH-cytochrome b5 reductase (b5R), is made. Although the clinical pictures of type I and II are strikingly different, mutations in the diaphorase (DIA1) gene located on chromosome 22 have been described in both types. In the present patient, two newly identified mutations, both leading to a stop codon in exon 4 (Gln77Ter) and in exon 6 (Arg160Ter), were found. Identification of different mutations at different positions in the DIA1 gene might shed light on the clinical and biochemical differences between methemoglobinemia type I and type II.

A Thai boy with hereditary enzymopenic methemoglobinemia type II.

Individuals with methemoglobin exceeding 1.5 g/dl have clinically obvious central cyanosis. Hereditary methemoglobinemia is due either to autosomal dominant M hemoglobins or to autosomal recessive enzymopenic methemoglobinemia. Four types of enzymopenic methemoglobinemia have been described. In addition to methemoglobinemia, individuals with type II, which is the generalized cytochrome b5 reductase deficiency, have severe and progressive neurological disabilities. Here we report a 3-year-old Thai boy with type II hereditary enzymopenic methemoglobinemia. He was born to a second-cousin couple. His central cyanosis was first observed around 10 months of age. His neurological abnormalities were seizures beginning at 1 year of age, microcephaly, and inability to hold his head up. His cardiovascular and pulmonary evaluations were unremarkable. Methemoglobin level by spectral absorption pattern was 18 per cent. A qualitative enzymatic assay confirmed the deficiency of the cytochrome b5 reductase enzyme. With this definite diagnosis, a prenatal diagnosis for the next child of this couple will be possible.

Four new mutations in the NADH-cytochrome b5 reductase gene from patients with recessive congenital methemoglobinemia type II.

Recessive congenital methemoglobinemia (RCM) due to NADH-cytochrome b5 reductase (cytb5r) deficiency leads to two different types of diseases. In the type I form, cyanosis is the only symptom, and the soluble enzyme is defective in red blood cells. In the type II form, cyanosis is associated with severe mental retardation and neurologic impairment; the enzymatic defect is systemic, involving both soluble and membrane-bound isoforms. We characterized mutations responsible for cytb5r deficiency in three unrelated patients with severe RCM type II. The first patient presented a homozygous exon 5 skipping. The only mutation detected was a homozygous G to C transversion at position +8, downstream from the 5' splice site of exon 5. We suggest that this unusual mutation might be responsible for the abnormal splicing of the primary transcripts, resulting in frameshift with premature STOP codon. The second mutation found corresponds to a homozygous C to T transition changing the Arg-218 codon to a premature STOP codon in exon 8. The third case was a compound heterozygote, carrying two different mutant alleles in the cyb5r gene. One allele presented a missense mutation with replacement of Cys-203 (TGC) by Arg (CGC) in exon 7. The second allele carried a 3-bp deletion (TGA) of nucleotides 815 to 817, modifying two contiguous codons in exon 9 of the cDNA with loss of Met-272. These results confirm the genetic polymorphism of cytb5r gene mutations identified in RCM type II, as observed for the mutations described in the RCM type I, and shed light on the molecular bases of the two different diseases associated with cytb5r deficiency.

Cytochrome b5 reductase deficiency, an uncommon cause of cyanosis.

A patient with cyanosis due to methaemoglobinaemia caused by cytochrome b5 reductase deficiency is described. Investigation of his family confirmed transmission of this disorder as an autosomal recessive trait. The consequences of this rare condition are discussed.

[Methemoglobinemias. Cytochrome b5-reductase deficiency]. / Metkhemoglobinemii. Tsitokhrom b5-reduktazna nedostatuchnost.

In the paper are considered the basic mechanisms, leading to accumulation of methemoglobin in the red blood cells. The inherited methemoglobinaemia, due to enzymatic failure of cytochrome B5-reductase is considered.

New variant of cytochrome b5 reductase deficiency (b5RKurashiki) in red cells, platelets, lymphocytes, and cultured fibroblasts with congenital methemoglobinemia, mental and neurological retardation, and skeletal anomalies.

A Japanese man with cytochrome b5 reductase (b5R) deficiency in various blood cell lineages (red cells, platelets, and lymphocytes) and in cultured fibroblasts demonstrated congenital methemoglobinemia associated with mental and neurological retardation, and various skeletal anomalies, such as spondylosis deformans and finger joint deformations, which have never been described in association with this enzyme deficiency. Cytochrome b5 reductase deficiency was most severe in red cells (0.3-4%) and less marked in platelets (13-27%), lymphocytes (18-31%), and fibroblasts (50%). The present case appears to be a new variant of cytochrome b5 reductase deficiency (b5RKurashiki).

Exonic point mutations in NADH-cytochrome B5 reductase genes of homozygotes for hereditary methemoglobinemia, types I and III: putative mechanisms of tissue-dependent enzyme deficiency.

We analyzed the NADH-cytochrome b5 reductase gene of hereditary methemoglobinemia type I and type III, by using PCR-related techniques. The mutation in type I is a guanine-to-adenine substitution in codon 57 of exon 3 of the NADH-cytochrome b5 reductase gene, and the sense of this codon is changed from arginine to glutamine. In type III the mutation is a thymine-to-cytosine transition in codon 148 of exon 5, causing leucine-to-proline replacement in type III. The former mutation abolishes the MspI recognition site. Homozygosity for the former mutation in a patient with type I was confirmed by restriction analysis of PCR-amplified fragments and by dot blot hybridization of amplified products with allele-specific oligonucleotide probes. The latter mutation generates a recognition site for MspI. Amplification of exon 5 by PCR followed by digestion with MspI revealed homozygosity for this mutation in patients with type-III. Putative mechanisms of tissue-dependent enzyme defects in hereditary methemoglobinemia are discussed.

Serine-proline replacement at residue 127 of NADH-cytochrome b5 reductase causes hereditary methemoglobinemia, generalized type.

Hereditary methemoglobinemia is an autosomal recessive disorder characterized by NADH-cytochrome b5 reductase (b5R) deficiency. In an attempt to clarify the molecular mechanisms involved in the enzyme deficiency, we isolated the b5R gene from a patient homozygous for hereditary methemoglobinemia, generalized type, and compared its nucleotide sequence with that of the normal NADH-cytochrome b5R gene. Only one difference was observed; a thymidine at the first position of codon 127 (TCT) was altered to a cytidine in the b5R gene of the patient, resulting in replacement of serine with proline. Dot blot hybridization of the amplified DNA samples with allele-specific oligonucleotide probes showed that the proband and her brothers were homozygous for this mutation and that their father was heterozygous. Although the activity of b5R in lymphoblastoid cells from homozygotes was reduced to 10% of the normal level, RNA blot and protein blot analyses of the lymphoblastoid cells showed that synthesis of b5R messenger RNA and the b5R polypeptide were normal. Serine at residue 127 is presumed to be in an alpha-helix structure that is part of a nucleotide-binding domain. These observations suggest that replacement of Pro-127 causes a significant conformation change in the nucleotide-binding domain that affects electron transport from NADH to cytochrome b5. Functional enzyme deficiency results in a generalized type of hereditary methemoglobinemia.

Tissue specific defect of complex I of the mitochondrial respiratory chain.

Deficiency of complex I is one of the most commonly reported defects of the mitochondrial respiratory chain in man. Clinical evidence of tissue specific expression of complex I deficiency has not previously been confirmed biochemically. We report here slow oxidation of NAD+-linked substrates, low activity of complex I and low amounts of immunoreactive complex I peptides in skeletal muscle mitochondria from a patient with muscle weakness and lactic acidosis. In liver mitochondria complex I activity was normal and all the immunoreactive subunits of complex I were present in normal amounts.