Transition cow nutrition and management strategies of dairy herds in the northeastern United States: Part I-Herd description and performance characteristics.

Our objective was to describe management and herd characteristics of the transition period on freestall dairy herds in the northeastern United States using an on-farm survey and prospective cohort design. Enrolled herds (n = 72) had a median of 900 milking cows (range: 345-2,900) and a rolling herd average of 12,674 kg (standard deviation ± 1,220 kg), and 87.2% (n = 82/94) of fresh pens were milked at least 3×/d. The prevalence of herds with ≥15% of sampled cows with elevated concentrations of nonesterified fatty acids prepartum (≥0.27 mmol/L, 2-14 d before parturition) and postpartum [primiparous: ≥0.60 mmol/L, multiparous: ≥0.70 mmol/L, 3-14 d in milk (DIM)], ß-hydroxybutyrate postpartum (≥1.2 mmol/L, 3-14 DIM), and haptoglobin postpartum (≥1 g/L, 0-12 DIM) was 51%, 51%, 51%, and 57%, respectively. In most herds, cows were moved to a calving pen when showing signs of labor (73.6%; n = 53/72) instead of 0 to 3 d before expected calving (26.4%, n = 19/72). Cows remained in the calving or maternity pen for a median (range) time of 2 (0-24) h after parturition before moving to the next pen. Primiparous cows remained in the first pen moved to after parturition for a longer period than multiparous cows [median (range) days: 12 (1.5-25) vs. 6 (1.5-22)]. Approximately 20% of herds had routine vaccinations administered in the maternity or calving pen, first pen after parturition, or both. Almost all herds (n = 69/72) performed fresh cow health checks; however, only 53% (n = 38/72) locked up all fresh cows daily. More herds housed primiparous and multiparous cows in separate pens during the far-off dry (65.3%; n = 47/72) and high-lactation (81.9%; n = 59/72) periods compared with the close-up dry (31.9%; n = 23/72) and fresh periods (27.8%; n = 20/72). At least half of the pens observed during the far-off dry, close-up dry, and fresh periods had a stocking density <100%. Approximately one-third of pens observed during the far-off dry period had feed pushed up ≤4×/d compared with approximately 15 to 20% of pens observed during the close-up dry, fresh, and high-lactation periods. More than half of the total mixed ration samples acquired from the far-off and close-up dry period visits had greater than the recommended proportion of particles in the 19-mm screen of the Penn State Particle Separator. The results of this observational study illustrated the range of management practices used in freestall herds in this region and lay the groundwork for future hypothesis-driven studies using this sampled population.

Recessive mutations in NDUFA2 cause mitochondrial leukoencephalopathy.

Deficiencies of mitochondrial respiratory chain complex I frequently result in leukoencephalopathy in young patients, and different mutations in the genes encoding its subunits are still being uncovered. We report 2 patients with cystic leukoencephalopathy and complex I deficiency with recessive mutations in NDUFA2, an accessory subunit of complex I. The first patient was initially diagnosed with a primary systemic carnitine deficiency associated with a homozygous variant in SLC22A5, but also exhibited developmental regression and cystic leukoencephalopathy, and an additional diagnosis of complex I deficiency was suspected. Biochemical analysis confirmed a complex I deficiency, and whole-exome sequencing revealed a homozygous mutation in NDUFA2 (c.134A>C, p.Lys45Thr). Review of a biorepository of patients with unsolved genetic leukoencephalopathies who underwent whole-exome or genome sequencing allowed us to identify a second patient with compound heterozygous mutations in NDUFA2 (c.134A>C, p.Lys45Thr; c.225del, p.Asn76Metfs*4). Only 1 other patient with mutations in NDUFA2 and a different phenotype (Leigh syndrome) has previously been reported. This is the first report of cystic leukoencephalopathy caused by mutations in NDUFA2.

Whole-exome sequencing is a valuable diagnostic tool for inherited peripheral neuropathies: Outcomes from a cohort of 50 families.

The inherited peripheral neuropathies (IPNs) are characterized by marked clinical and genetic heterogeneity and include relatively frequent presentations such as Charcot-Marie-Tooth disease and hereditary motor neuropathy, as well as more rare conditions where peripheral neuropathy is associated with additional features. There are over 250 genes known to cause IPN-related disorders but it is estimated that in approximately 50% of affected individuals a molecular diagnosis is not achieved. In this study, we examine the diagnostic utility of whole-exome sequencing (WES) in a cohort of 50 families with 1 or more affected individuals with a molecularly undiagnosed IPN with or without additional features. Pathogenic or likely pathogenic variants in genes known to cause IPN were identified in 24% (12/50) of the families. A further 22% (11/50) of families carried sequence variants in IPN genes in which the significance remains unclear. An additional 12% (6/50) of families had variants in novel IPN candidate genes, 3 of which have been published thus far as novel discoveries (KIF1A, TBCK, and MCM3AP). This study highlights the use of WES in the molecular diagnostic approach of highly heterogeneous disorders, such as IPNs, places it in context of other published neuropathy cohorts, while further highlighting associated benefits for discovery.

Debunking Occam's razor: Diagnosing multiple genetic diseases in families by whole-exome sequencing.

BACKGROUND: Recent clinical whole exome sequencing (WES) cohorts have identified unanticipated multiple genetic diagnoses in single patients. However, the frequency of multiple genetic diagnoses in families is largely unknown. AIMS: We set out to identify the rate of multiple genetic diagnoses in probands and their families referred for analysis in two national research programs in Canada. MATERIALS & METHODS: We retrospectively analyzed WES results for 802 undiagnosed probands referred over the past 5 years in either the FORGE or Care4Rare Canada WES initiatives. RESULTS: Of the 802 probands, 226 (28.2%) were diagnosed based on mutations in known disease genes. Eight (3.5%) had two or more genetic diagnoses explaining their clinical phenotype, a rate in keeping with the large published studies (average 4.3%; 1.4 - 7.2%). Seven of the 8 probands had family members with one or more of the molecularly diagnosed diseases. Consanguinity and multisystem disease appeared to increase the likelihood of multiple genetic diagnoses in a family. CONCLUSION: Our findings highlight the importance of comprehensive clinical phenotyping of family members to ultimately provide accurate genetic counseling.

Synovial chemokine expression and relationship with knee symptoms in patients with meniscal tears.

OBJECTIVE: In patients with knee OA, synovitis is associated with knee pain and symptoms. We previously identified synovial mRNA expression of a set of chemokines (CCL19, IL-8, CCL5, XCL-1, CCR7) associated with synovitis in patients with meniscal tears but without radiographic OA. CCL19 and CCR7 were also associated with knee symptoms. This study sought to validate expression of these chemokines and association with knee symptoms in more typical patients presenting for meniscal arthroscopy, many who have pre-existing OA. DESIGN: Synovial fluid (SF) and biopsies were collected from patients undergoing meniscal arthroscopy. Synovial mRNA expression was measured using quantitative RT-PCR. The Knee Injury and Osteoarthritis Outcome Score (KOOS) was administered preoperatively. Regression analyses determined if associations between chemokine mRNA levels and KOOS scores were independent of other factors including radiographic OA. CCL19 in SF was measured by ELISA, and compared to patients with advanced knee OA and asymptomatic organ donors. RESULTS: 90% of patients had intra-operative evidence of early cartilage degeneration. CCL19, IL-8, CCL5, XCL1, CCR7 transcripts were detected in all patients. Synovial CCL19 mRNA levels independently correlated with KOOS Activities of Daily Living (ADL) scores (95% CI [-8.071, -0.331], P = 0.036), indicating higher expression was associated with more knee-related dysfunction. SF CCL19 was detected in 7 of 10 patients, compared to 4 of 10 asymptomatic donors. CONCLUSION: In typical patients presenting for meniscal arthroscopy, synovial CCL19 mRNA expression was associated with knee-related difficulty with ADL, independent of other factors including presence of radiographic knee OA.

Whole-exome sequencing broadens the phenotypic spectrum of rare pediatric epilepsy: a retrospective study.

Whole-exome sequencing (WES) has transformed our ability to detect mutations causing rare diseases. FORGE (Finding Of Rare disease GEnes) and Care4Rare Canada are nation-wide projects focused on identifying disease genes using WES and translating this technology to patient care. Rare forms of epilepsy are well-suited for WES and we retrospectively selected FORGE and Care4Rare families with clinical descriptions that included childhood-onset epilepsy or seizures not part of a recognizable syndrome or an early-onset encephalopathy where standard-of-care investigations were unrevealing. Nine families met these criteria and a diagnosis was made in seven, and potentially eight, of the families. In the eight families we identified mutations in genes associated with known neurological and epilepsy disorders: ASAH1, FOLR1, GRIN2A (two families), SCN8A, SYNGAP1 and SYNJ1. A novel and rare mutation was identified in KCNQ2 and was likely responsible for the benign seizures segregating in the family though additional evidence would be required to be definitive. In retrospect, the clinical presentation of four of the patients was considered atypical, thereby broadening the phenotypic spectrum of these conditions. Given the extensive clinical and genetic heterogeneity associated with epilepsy, our findings suggest that WES may be considered when a specific gene is not immediately suspected as causal.

First Report of Soybean Cyst Nematode (Heterodera glycines Ichinohe) on Soybean in the Province of Quebec, Canada.

In eastern Canada, soybean, Glycine max (L.) Merr., is the most important legume, and its cultivation is expanding to new regions as cultivars for the short growing season are developed. The soybean cyst nematode (SCN), Heterodera glycines Ichinohe, is among the most destructive pests of soybean in the world. This nematode is also under quarantine regulations in many countries, including Canada. Until now, in Canada, SCN was only reported in the province of Ontario. Since its first detection in 1988 in the southwestern part of the province (1), SCN has been found in 12 other counties. It appears that SCN has been spreading in a north and northeast direction along the St. Lawrence River. We report here the first detection of SCN in the province of Quebec. Second stage juveniles (J2) and cysts were found in St. Anicet, Quebec, Canada, in a 10-ha soybean field. Light textured soil is a characteristic of the field, the same site where Pratylenchus alleni was recently discovered (2) and where irregular patches of stunted soybean plants were observed. Morphological and molecular studies of J2 and cysts confirmed the identification of this nematode population as SCN. The J2 were typical for SCN with a body length of 393 to 428 µm, lateral fields harboring four straight lines, a well-developed stylet 23 to 25 µm long, sub-ventral base knobs with posterior slops, a tail length of 43 to 50 µm, and a hyaline part of 23 to 29 µm. Cysts were brown and lemon-shaped with a posterior protuberance, ambifenestrated, underbridged, and had a strongly developed bullae. Key morphometrics were: a cyst fenestra 40 to 57 µm long and 28 to 44 µm wide, and a vulval slit 39 to 53 µm long. All of these are coincident with those of SCN (3). Ribosomal DNA of the ITS, 18S, and D2/D3 regions, and mitochondrial COX1 gene were PCR amplified from cysts and J2s gDNA using primers ITS-F (5'-TTGATTACGTCCCTGCCCTTT-3') and ITS-R (5'-ACGAGCCGAGTGATCCACCG-3'); 18S-F (5'-TTGGATAACTGTGGTTTAACTAG-3') and 18S-R (5'-ATTTCACCTCTCACGCAACA-3'); D2A (5'-ACAAGTACCGTGAGGGAAAGT-3') and D3B (5'-GACCCGTCTTGAAACACGGA-3'); and COXI-F (5'-CCTACTATGATTGGTGGTTTTGGTAATTG-3') and COX1-R (5'-GTAGCAGCAGTAAAATAAGCACG-3'), respectively, and sequenced. The nucleotide sequences were 98 to 100% similar to those of SCN found in NCBI nr database (July 2013). All the sequences have been submitted to GenBank with the following accession numbers: ITS (KF453621); 18S (KF453622); D2/D3 (KF453623); and COX1 (KF453624). Using species specific sequence characterized amplified region (SCAR) primers (4) also confirmed this was H. glycines. This is the first reported case of SCN in Quebec, Canada. The proximity of St. Anicet to the Ontario border is in accordance with the North/Northeastern dispersal hypothesis. The HG type of the SCN population will have to be determined before any resistant cultivars are deployed for the management of this pathogen in the province. References: (1) T. R. Anderson et al. Plant Dis. 72:453, 1988. (2) G. Bélair et al. Plant Dis. 97:292, 2013. (3) R. H. Mulvey. Can. J. Zool. 50:1277, 1972. (4) S. Ou et al. Nematology 10:397, 2008.

Diversity of ARSACS mutations in French-Canadians.

BACKGROUND: The growing number of spastic ataxia of Charlevoix-Saguenay (SACS) gene mutations reported worldwide has broadened the clinical phenotype of autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS). The identification of Quebec ARSACS cases without two known SACS mutation led to the development of a multi-modal genomic strategy to uncover mutations in this large gene and explore phenotype variability. METHODS: Search for SACS mutations by combining various methods on 20 cases with a classical French-Canadian ARSACS phenotype without two mutations and a group of 104 sporadic or recessive spastic ataxia cases of unknown cause. Western blot on lymphoblast protein from cases with different genotypes was probed to establish if they still expressed sacsin. RESULTS: A total of 12 mutations, including 7 novels, were uncovered in Quebec ARSACS cases. The screening of 104 spastic ataxia cases of unknown cause for 98 SACS mutations did not uncover carriers of two mutations. Compounds heterozygotes for one missense SACS mutation were found to minimally express sacsin. CONCLUSIONS: The large number of SACS mutations present even in Quebec suggests that the size of the gene alone may explain the great genotypic diversity. This study does not support an expanding ARSACS phenotype in the French-Canadian population. Most mutations lead to loss of function, though phenotypic variability in other populations may reflect partial loss of function with preservation of some sacsin expression. Our results also highlight the challenge of SACS mutation screening and the necessity to develop new generation sequencing methods to ensure low cost complete gene sequencing.

Founder mutation for α-sarcoglycan-LGMD2D in a Magdalen Islands Acadian cluster.

BACKGROUND: We have recruited a group of four living and reviewed the records of six deceased distantly related French-Canadians of Acadian descent affected by a childhood-onset form of recessive limb-girdle muscular dystrophy (LGMD). All cases originate from the small archipelago of the Magdalen Islands (population: 13,000) isolated in the Gulf of St-Lawrence. METHODS: Based on the likely sharing of the same founder mutation we completed a 319K SNPs genome-wide scan to identify the disease locus and then screen candidate genes in this region. RESULTS: All patients had normal initial motor milestones. They presented with limb girdle weakness at the average age of seven years (5-11). Progressive weakness led to loss of ambulation at a wide range of ages (10-39). Patients also developed macroglossia, large calves and mild to moderate contractures, hyperlordosis and decreased pulmonary function. Creatine kinase levels were elevated (1,800-10,000 U/L) in the first decades, but decreased with progression of disease. Homozygosity mapping uncovered a shared chromosomal region of 6.33Mb. The alpha sarcoglycan (SGCA) gene, mutated in LGMD2D, lay in this candidate interval. Sequencing of all SGCA exons uncovered a shared homozygous missense mutation (c. 229C>T, p.R77C), the most common SGCA mutation internationally reported. Using demographic data, we estimated a high carrier rate of 1/22. CONCLUSION: The p.R77C mutation has also been observed in many populations, including in France and Spain (Basques). This corresponds to the first reported recessive founder disease for the Magdalen Islands, an archipelago settled in the XIXth century, largely by Acadian immigrants.

A novel cyclic nucleotide-gated ion channel enriched in synaptic terminals of isotocin neurons in zebrafish brain and pituitary.

Cyclic nucleotide-gated (CNG) channels are nonselective cation channels opened by binding of intracellular cyclic GMP or cyclic AMP. CNG channels mediate sensory transduction in the rods and cones of the retina and in olfactory sensory neurons, but in addition, CNG channels are also expressed elsewhere in the CNS, where their physiological roles have not yet been well defined. Besides the CNG channel subtypes that mediate vision and olfaction, zebrafish has an additional subtype, CNGA5, which is expressed almost exclusively in the brain. We have generated CNGA5-specific monoclonal antibodies, which we use here to show that immunoreactivity for CNGA5 channels is highly enriched in synaptic terminals of a discrete set of neurons that project to a subregion of the pituitary, as well as diffusely in the brain and spinal cord. Double labeling with a variety of antibodies against pituitary hormones revealed that CNGA5 is located in the terminals of neuroendocrine cells that secrete the nonapeptide hormone/transmitter isotocin in the neurohypophysis, brain, and spinal cord. Furthermore, we show that CNGA5 channels expressed in Xenopus oocytes are highly permeable to Ca(2+), which suggests that the channels are capable of modulating isotocin release in the zebrafish brain and pituitary. Isotocin is the teleost homolog of the mammalian hormone oxytocin, and like oxytocin, it regulates reproductive and social behavior. Therefore, the high calcium permeability of CNGA5 channels and their strategic location in isotocin-secreting synaptic terminals suggest that activation of CNGA5 channels in response to cyclic nucleotide signaling may have wide-ranging neuroendocrine and behavioral effects.

A novel founder SCN4A mutation causes painful cold-induced myotonia in French-Canadians.

BACKGROUND: Myotonia is observed in classic congenital myotonia caused by CLCN1 mutations and in sodium-channel myotonia (SCM) due to SCN4A mutations. METHODS: We assessed 66 electrically proven cases of myotonia belonging to 17 French-Canadian families living in the Saguenay Lac St-Jean area of Quebec, a region well known for its genetic founder effects. The CLCN1 gene was sequenced in one affected member of each family. SCN4A exons with known SCM mutations were subsequently sequenced in families where no CLCN1 mutations were found. RESULTS: Six families, 33% of cases (22/66), presenting classic congenital myotonia phenotypes were found to carry two previously identified CLCN1 mutations. In the other 11 families comprising 66% of cases (44/66), a new dominant SCN4A mutation in exon 24 (M1476I) was uncovered and segregated with a variable SCM phenotype. Although all carriers of this novel mutation had electrical myotonia, some were asymptomatic (25%) and age at onset was variable in the others (5 to 67, mean 21). Cold aggravated myotonia was observed in 41% of cases and painful myotonia in 18%. Additional features observed include aggravation of symptoms with pregnancies (7%), localized muscle swelling (2%), myotonic reactions to anesthesia (2%), and food-induced paralysis (2%). CONCLUSIONS: This cohort is the largest described with a variable sodium-channel myotonia phenotype caused by a single SCN4A mutation. The clinical variability observed in this cohort underlines the phenotypic heterogeneity of SCN4A mutations and suggests that variants in other genes likely modulate clinical expression.

A novel autosomal recessive limb-girdle muscular dystrophy with quadriceps atrophy maps to 11p13-p12.

Limb-girdle muscular dystrophies (LGMD) are a heterogeneous group of pathologies. We have identified a cohort of 14 French-Canadian patients from eight different families displaying a novel form of LGMD with an autosomal recessive inheritance. These patients share some features with previously described cases of 'quadriceps myopathy' that evolved into an LGMD. All demonstrate quadriceps femoris asymmetrical atrophy. Creatine kinase values were variable from normal to 6000 U/l. Clinical evaluations and MRI studies demonstrate a variable intrafamilial and interfamilial phenotype. Asymmetrical muscle involvement was clinically observed and confirmed by imaging. MRI studies suggest that the hamstrings and the adductor magnus are the first limb muscles to demonstrate fatty infiltration. Muscle pathology shows no sign of active inflammation but increased endomysial connective tissue associated with basal lamina duplication and collagen disorganization. A genome-wide scan using the two largest families uncovered linkage to marker D11S1360 on chromosome 11p12 [multipoint logarithm of the odds (LOD) score of 2.78]. Further genotyping for the eight families confirmed linkage to this new LGMD locus (multipoint LOD score of 4.56). Fine mapping subsequently defined a less than 3.3 cM candidate interval on 11p13-p12. Haplotype analysis of carrier chromosomes suggests that the most frequent mutation may account for up to 81.3% of French-Canadian mutations. In this study, we describe the chromosomal locus of a new form of recessive LGMD with prominent quadriceps femoris atrophy.

A new form of congenital muscular dystrophy with joint hyperlaxity maps to 3p23-21.

Congenital muscular dystrophies (CMDS) are a heterogeneous group of disorders. A growing number of CMDS have been found to be associated with joint hyperlaxity. We recruited 14 French-Canadian cases belonging to 11 families affected by a novel autosomal recessive congenital muscular dystrophy with hyperlaxity (CMDH). All cases come from the southwestern part of Quebec, suggesting a new French-Canadian founder effect. All patients present muscle weakness, proximal contractures coexisting with distal joint hyperlaxity. Pathological and genetic studies have excluded that mutations in the three genes coding for collagen VI subunits are responsible for this disease. A genome-wide scan established linkage of two CMDH families to a region on chromosome 3p23-21. Further linkage analysis confirmed that all families are linked to the same region (log of the odds score of 5.3). Haplotype analysis defines a 1.6-cM candidate interval and suggests that two common mutations may account for 78% of carrier chromosomes. This study describes and maps a new form of recessive CMD with joint hyperlaxity distinct from Ullrich and Bethlem myopathies with a founder effect in the French-Canadian population.

A new autosomal recessive spastic ataxia associated with frequent white matter changes maps to 2q33-34.

Recessive ataxias are a heterogeneous group of diseases. We identified a group of 23 French-Canadian cases belonging to 17 families affected by an autosomal recessive spastic ataxia associated with frequent white matter changes. The fact that 59% of these families have a genealogical relationship to the Portneuf County of Quebec suggests that this is a new form of ataxia with a regional founder effect. All cases present with cerebellar ataxia and spasticity. There is great intrafamilial and interfamilial variability, as illustrated by the spectrum of age of diagnosis (range: 2-59 years, mean: 15.0) and the presence of white matter changes on MRI in 52.4% of cases. The more severe cases have spasticity from birth, scoliosis, dystonia and cognitive impairment and were considered cases of cerebral palsy. Brain MRI constantly shows cerebellar atrophy, which in some cases may be associated with cortical atrophy, leucoencephalopathy and corpus callosum thinning. A genome wide scan uncovered linkage of three families to marker D2S2321 localized on chromosome 2q33-34. Linkage analysis confirmed that all families are linked to the same region [multipoint log of the odds (LOD) score of 5.95]. Haplotype analysis and allele sharing suggest that one common mutation may account for 97% of carrier chromosomes in Quebec. The uncovering of the mutated gene may point to a common pathway for pyramidal and cerebellar degeneration as both are often observed in recessive ataxias and complicated paraplegias.

Electrophysiological characteristics of limbic and motor globus pallidus internus (GPI) neurons in two cases of Lesch-Nyhan syndrome.

OBJECTIVE: Lesch-Nyhan syndrome is a rare and debilitating condition characterized by dystonia and self-mutilating behavior. In order to shed light on the pathophysiology of dystonia, we report the pallidal electrophysiological activity recorded in two patients during deep brain stimulation surgery (DBS). METHODS: Microrecordings were performed on 162 neurons along four tracks aimed at the right and left anterior (limbic) and posterior (motor) globus pallidus internus (GPI). RESULTS: Regardless of the anesthetic agent used (propofol or sevoflurane), both patients showed similar neurons firing rates in the four regions studied, namely the limbic and motor portions of the globus pallidus externus (GPE) or GPI. In both patients, firing rates were similar in the GPE (12.2+/-1.8 Hz, N=38) and GPI (13.2+/-1.0 Hz, N=83) portions of the limbic track, while the motor GPE fired at a higher frequency (23.8+/-2.7 Hz, N=18) than the motor GPI (12.5+/-1.4 Hz, N=23). CONCLUSIONS: These results demonstrate that light propofol or sevoflurane anesthesia influences pallidal activity in a similar way. Electrophysiological recordings suggest that Lesch-Nyhan syndrome might be characterized by analogous firing frequencies in the limbic GPE and GPI while motor GPE would tend to fire at higher rate than the motor GPI. It is therefore tempting to suggest that the symptoms that are observed in Lesch-Nyhan syndrome might result from motor GPI inhibition. SIGNIFICANCE: This observation may confirm the Albin and Delong's model of the basal nuclei in hypokinetic and hyperkinetic disorders.

Double mutant studies identify electrostatic interactions that are important for docking cytochrome c2 onto the bacterial reaction center.

Cytochrome c2 (cyt) is the mobile electron donor to the reaction center (RC) in photosynthetic bacteria. The electrostatic interactions involved in the dynamics of docking of cyt onto the RC were examined by double mutant studies of the rates of electron transfer between six modified Rhodobacter sphaeroides RCs in which negatively charged acid residues were replaced with Lys and five modified Rhodobacter capsulatus Cyt c2 molecules in which positively charged Lys residues were replaced with Glu. We measured the second-order rate constant, k2, for electron transfer from the reduced cyt to the oxidized primary donor on the RC, which reflects the energy of the transition state for the formation of the active electron transfer complex. Strong interactions were found between Lys C99 and Asp M184/Glu M95, and between Lys C54 and Asp L261/Asp L257. The interacting residues were found to be located close to each other in the recently determined crystal structure of the cyt-RC complex [Axelrod, H., et al. (2002) J. Mol. Biol. (in press)]. The interaction energies were approximately inversely proportional to the distances between charges. These results support earlier suggestions [Tetreault, M., et al. (2001) Biochemistry 40, 8452-8462] that the structure of the transition state in solution resembles the structure of the cyt-RC complex in the cocrystal and indicate that specific electrostatic interactions facilitate docking of the cyt onto the RC in a configuration optimized for both binding and electron transfer. The specific interaction between Asp M184 and Lys C99 may help to nucleate short-range hydrophobic contacts.

Interaction between cytochrome c2 and the photosynthetic reaction center from Rhodobacter sphaeroides: effects of charge-modifying mutations on binding and electron transfer.

The electrostatic interactions governing binding and electron transfer from cytochrome c(2) (cyt c(2)) to the reaction center (RC) from the photosynthetic bacteria Rhodobacter sphaeroides were studied by using site-directed mutagenesis to change the charges of residues on the RC surface. Charge-reversing mutations (acid --> Lys) decreased the binding affinity for cyt c(2). Dissociation constants, K(D) (0.3--250 microM), were largest for mutations of Asp M184 and nearby acid residues, identifying the main region for electrostatic interaction with cyt c(2). The second-order rate constants, k(2) (1--17 x 10(8) M(-1) s(-1)), increased with increasing binding affinity (log k(2) vs log 1/K(D) had a slope of approximately 0.4), indicating a transition state structurally related to the final complex. In contrast, first-order electron transfer rates, k(e), for the bound cyt did not change significantly (<3-fold), indicating that electron tunneling pathways were unchanged by mutation. Charge-neutralizing mutations (acid --> amide) showed changes in binding free energies of approximately 1/2 the free energy changes due to the corresponding charge-reversing mutations, suggesting that the charges in the docked complex remain well solvated. Charge-enhancing mutations (amide --> acid) produced free energy changes of the same magnitude (but opposite sign) as changes due to the charge-neutralizing mutations in the same region, indicating a diffuse electrostatic potential due to cyt c(2). A two-domain model is proposed, consisting of an electrostatic docking domain with charged surfaces separated by a water layer and a hydrophobic tunneling domain with atomic contacts that provide an efficient pathway for electron transfer.