Biomedical subjects

H Mei

Publications and source records attributed to H Mei.

At least 19 recordsLinked to original sources

PubMed2006

An analysis paradigm for investigating multi-locus effects in complex disease: examination of three GABA receptor subunit genes on 15q11-q13 as risk factors for autistic disorder.

Gene-gene interactions are likely involved in many complex genetic disorders and new statistical approaches for detecting such interactions are needed. We propose a multi-analytic paradigm, relying on convergence of evidence across multiple analysis tools. Our paradigm tests for main and interactive effects, through allele, genotype and haplotype association. We applied our paradigm to genotype data from three GABAA receptor subunit genes (GABRB3, GABRA5, and GABRG3) on chromosome 15 in 470 Caucasian autism families. Previously implicated in autism, we hypothesized these genes interact to contribute to risk. We detected no evidence of main effects by allelic (PDT, FBAT) or genotypic (genotype-PDT) association at individual markers. However, three two-marker haplotypes in GABRG3 were significant (HBAT). We detected no significant multi-locus associations using genotype-PDT analysis or the EMDR data reduction program. However, consistent with the haplotype findings, the best single locus EMDR model selected a GABRG3 marker. Further, the best pairwise genotype-PDT result involved GABRB3 and GABRG3, and all multi-locus EMDR models also selected GABRB3 and GABRG3 markers. GABA receptor subunit genes do not significantly interact to contribute to autism risk in our overall data set. However, the consistency of results across analyses suggests that we have defined a useful framework for evaluating gene-gene interactions.

Autistic Disorder↗

PubMed2005

Identification of significant association and gene-gene interaction of GABA receptor subunit genes in autism.

Autism is a common neurodevelopmental disorder with a significant genetic component. Existing research suggests that multiple genes contribute to autism and that epigenetic effects or gene-gene interactions are likely contributors to autism risk. However, these effects have not yet been identified. Gamma-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the adult brain, has been implicated in autism etiology. Fourteen known autosomal GABA receptor subunit genes were studied to look for the genes associated with autism and their possible interactions. Single-nucleotide polymorphisms (SNPs) were screened in the following genes: GABRG1, GABRA2, GABRA4, and GABRB1 on chromosome 4p12; GABRB2, GABRA6, GABRA1, GABRG2, and GABRP on 5q34-q35.1; GABRR1 and GABRR2 on 6q15; and GABRA5, GABRB3, and GABRG3 on 15q12. Intronic and/or silent mutation SNPs within each gene were analyzed in 470 white families with autism. Initially, SNPs were used in a family-based study for allelic association analysis--with the pedigree disequilibrium test and the family-based association test--and for genotypic and haplotypic association analysis--with the genotype-pedigree disequilibrium test (geno-PDT), the association in the presence of linkage (APL) test, and the haplotype family-based association test. Next, with the use of five refined independent marker sets, extended multifactor-dimensionality reduction (EMDR) analysis was employed to identify the models with locus joint effects, and interaction was further verified by conditional logistic regression. Significant allelic association was found for markers RS1912960 (in GABRA4; P = .01) and HCV9866022 (in GABRR2; P = .04). The geno-PDT found significant genotypic association for HCV8262334 (in GABRA2), RS1912960 and RS2280073 (in GABRA4), and RS2617503 and RS12187676 (in GABRB2). Consistent with the allelic and genotypic association results, EMDR confirmed the main effect at RS1912960 (in GABRA4). EMDR also identified a significant two-locus gene-gene effect model involving RS1912960 in GABRA4 and RS2351299 in GABRB1. Further support for this two-locus model came from both the multilocus geno-PDT and the APL test, which indicated a common genotype and haplotype combination positively associated with disease. Finally, these results were also consistent with the results from the conditional logistic regression, which confirmed the interaction between GABRA4 and GABRB1 (odds ratio = 2.9 for interaction term; P = .002). Through the convergence of all analyses, we conclude that GABRA4 is involved in the etiology of autism and potentially increases autism risk through interaction with GABRB1. These results support the hypothesis that GABA receptor subunit genes are involved in autism, most likely via complex gene-gene interactions.

Autistic Disorder↗

PubMed2003

Expression array annotation using the BioMediator biological data integration system and the BioConductor analytic platform.

This paper presents the implementation of a model for expression array annotation (EAA) using the BioMediator biological data integration system along with BioConductor, an analytic tools platform. The model presented addresses the need for annotation sources identified during BioConductor inverted exclamation mark s development. Annotation provides us with well-curated genomic background knowledge for expression array analysis and interpretation. Annotation requests are constructed and posted to the query interface of the EAA package (the EAA model implemented as a component of BioConductor). The software enumerates all possible annotation paths for queries. These are then transformed to PQL queries and processed by BioMediator. Annotation entities returned from the EAA package answer the annotation request.

Computational Biology↗

PubMed2001

Quantitative screening and matrix effect studies of drug discovery compounds in monkey plasma using fast-gradient liquid chromatography/tandem mass spectrometry.

A higher-throughput bioanalytical method based on fast-gradient (1 min run time) high-performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS) was developed for screen-type analyses of plasma samples from early drug discovery studies in support of exploratory pharmacodynamic studies. The HPLC system equipped with minibore column was interfaced with either atmospheric pressure chemical ionization (APCI) or electrospray (ESI) ionization techniques. The matrix ion suppression effect of both quantitative HPLC/MS/MS analyses was compared using the post-column infusion system. The use of the described methods provided advantages such as a shorter chromatographic region of ion suppression, less solvent consumption and shorter run times in comparison with standard analytical column HPLC/MS/MS methods. The analytical results obtained by both HPLC/MS/MS methods were in good agreement (within 15% of error) and displayed a good correlation with the pharmacodynamic outcome.

2-Hydroxypropyl-beta-cyclodextrin↗

PubMed2001

If taken 1 hour before indinavir (IDV), didanosine does not affect IDV exposure, despite persistent buffering effects.

Concurrent administration of indinavir and didanosine significantly reduces the level of exposure to indinavir, but it is unclear how soon after didanosine administration indinavir may be given safely. We compared indinavir pharmacokinetics and gastric pH in 12 human immunodeficiency virus-positive patients by use of 800 mg of indinavir alone versus 800 mg of indinavir administered 1 h after didanosine administration. Median gastric pH was significantly higher when indinavir was taken after didanosine administration; however, no significant difference in the maximum concentration in plasma or the area under the concentration-time curve from time zero to 8 h was observed. Indinavir may be taken with a light meal 1 h following the administration of 400 mg of didanosine.

Adolescent↗

PubMed2001

[Clinical study of detenia cecal-ascending colon continent cutaneous urinary reservoir].

OBJECTIVE: To construct a good continent urinary diversion which is easy to be performed and has a low incidence of complications. METHODS: 26 cases of bladder cancer were given radical cystectomy before the cecal-ascending colon was excluded and 15-20 cm of the cecal-ascending colon was isolated. The colon teniae were then incised at the interval of 0.5-1.0 cm to construct the detenia cecal-ascending colon continent urinary diversion open to the umbilicus. RESULTS: All the patients were followed up for 21.1 +/- 10.1 months. Reliable continence was achieved in all with a low incidence of complications. The capacity of the reservoirs reached 350-600 ml 6 month after operation. Self catherizations were carried out every 3 to 6 hours, Urodynamic data showed a mean maximum filling pressure of 58.7 +/- 24.5 cmH2O, and a mean maximum urethral (efferent) closure pressure of 104.3 +/- 33.8 cmH2O. CONCLUSION: Detenia cecal-ascending colon continent urinary diversion is an ideal method.

Adult↗

PubMed2000

[Study of CFTR gene mutation in Chinese CUAVD patients].

OBJECTIVE: To analyze the frequency and hot spots of cystic fibrosis transmembrane conductance regulator(CFTR) gene in Chinese congenital unilateral absence of the vas deferens (CUAVD) patients. METHODS: The mutation of CFTR exons 2, 3, 4, 5, 6a, 8, 10, 11, 12, 13, 15A, 17b, 19A, 20, 21, and 23 were detected. PCR-single strand conformation polymorphism(SSCP) and direct sequencing were performed on 15 cases of Chinese CUAVD. RESULTS: One case exhibited an abnormal shift SSCP band in exon 17b of CFTR gene and subsequent DNA sequencing showed C to A transversion at position 3295 that led to a predicted change of Leusine(codon 1055, CTT) to Isoleucine(codon ATT). CONCLUSION: CFTR mutation could be detected in Chinese CUAVD patients. The missense mutation, Leu1055Ile, was identified as a novel CFTR mutation. It is necessary that Chinese CUAVD patients and their wives should be screened for CFTR gene prior to intracytoplasmic sperm injection.

Cystic Fibrosis Transmembrane Conductance Regulato↗

PubMed2000

KAI1/CD82 gene expression in benign prostatic hyperplasia and late-stage prostate cancer in Chinese.

AIM: To evaluate KAI1/CD82 expression in Chinese patients with benign prostatic hyperplasia (BPH) and late-stage carcinoma of prostate (CaP). METHODS: Thirty Chinese patients with benign prostatic hyperplasia and 34 with CaP (adenocarcinoma clinical stage C and D) were analyzed by means of immunohistochemical methods. RESULTS: The KAI1/CD82 expression in BPH tissue was all positive, which was uniformly located on the glandular cell membrane at the cell-to-cell borders, but KAI1/CD82 expression in metastasis CaP tissues was either significantly lower than that of BPH or negative, and the immunostaining pattern was not continuous. In late-stage CaP KAI1/CD82 expression was correlated inversely to the pathological grade ( P < 0.05), but not to clinical stage ( P > 0.05). CONCLUSION: The authors believe that decreased and negative KAI1/CD82 expression in late-stage CaP may be related to tumor progression and metastasis, and appears to be a prognostic marker.

Adenocarcinoma↗

PubMed2000

[Telomerase activity and cellular apoptosis of bladder cancers].

OBJECTIVE: To explore the relationship between clinicobiological acting, telomerase activity and cellular apoptosis of bladder cancer. METHODS: The intensity of telomerase activity and cellular apoptosis of bladder cancer were detected by telomeric repeat amplification protocol (TRAP) and terminal deoxynucleotidyl-transferase mediated dUTP-biotin nick end labeling (TUNEL) assay respectively. RESULTS: The positive rate of telomerase activity of bladder cancer was 89.29% and the apoptosis index of bladder cancer was (45.16 +/- 14.43)%. The intensity of telomerase activity and apoptosis index of bladder cancer were not related to patient's age, sex, size or number of tumor (P > 0.05), but to the "grade", "stage" and prognosis of bladder cancer (P < 0.01). The higher the intensity of telomerase activity or less cellular apoptosis, the poor prognosis of differentiated or more advanced bladder cancer. The intensity of telomerase activity was negatively correlated with cellular apoptosis of bladder cancer (r = -0.69, P < 0.01). CONCLUSIONS: The intensity of telomerase activity and cellular apoptosis are correlated with the grade, stage and prognosis of bladder cancer. The detection of telomerase activity and cellular apoptosis are helpful in clinical analysis and evaluation of prognosis of bladder cancer.

Adult↗

PubMed2000

[Intracytoplasmic injection with sperm from ejaculation, epididymis and testis in treating different male infertility with oligo-asthenozoospermia or azoospermia ].

OBJECTIVE: To retrospectively study 400 intracytoplasmic sperm injection (ICSI) cycles by using three different sources of sperm. METHOD: Different male infertility with oligo-asthenozoospermia or azoospermia was treated by ICSI using sperm from ejaculation, epididymis and testis. RESULTS: In the ejaculation group (n = 277), the fertilization rate was 69.6% and the clinical pregnancy rate 28.9%. In the 104 cycles using epididymal sperm, the fertilization and clinical pregnancy rate was 65.9% and 37.5% respectively. And the fertilization and clinical pregnancy of testis sperm (n = 19) was 65.7% and 21.1%. The difference of fertilization and clinical pregnancy rate among the three groups were not significant. CONCLUSIONS: Male infertility of different causes can be treated by ICSI. The outcome has nothing to do with the common parameter of semen except that the sperm motility is 0, but the effectiveness of ICSI should be studied by long-term observation.

Adult↗

PubMed1999

Role of configurational gating in intracomplex electron transfer from cytochrome c to the radical cation in cytochrome c peroxidase.

Electron transfer within complexes of cytochrome c (Cc) and cytochrome c peroxidase (CcP) was studied to determine whether the reactions are gated by fluctuations in configuration. Electron transfer in the physiological complex of yeast Cc (yCc) and CcP was studied using the Ru-39-Cc derivative, in which the H39C/C102T variant of yeast iso-1-cytochrome c is labeled at the single cysteine residue on the back surface with trisbipyridylruthenium(II). Laser excitation of the 1:1 Ru-39-Cc-CcP compound I complex at low ionic strength results in rapid electron transfer from RuII to heme c FeIII, followed by electron transfer from heme c FeII to the Trp-191 indolyl radical cation with a rate constant keta of 2 x 10(6) s-1 at 20 degrees C. keta is not changed by increasing the viscosity up to 40 cP with glycerol and is independent of temperature. These results suggest that this reaction is not gated by fluctuations in the configuration of the complex, but may represent the elementary electron transfer step. The value of keta is consistent with the efficient pathway for electron transfer in the crystalline yCc-CcP complex, which has a distance of 16 A between the edge of heme c and the Trp-191 indole [Pelletier, H., and Kraut, J. (1992) Science 258, 1748-1755]. Electron transfer in the complex of horse Cc (hCc) and CcP was examined using Ru-27-Cc, in which hCc is labeled with trisbipyridylruthenium(II) at Lys-27. Laser excitation of the Ru-27-Cc-CcP complex results in electron transfer from RuII to heme c FeII with a rate constant k1 of 2.3 x 10(7) s-1, followed by oxidation of the Trp-191 indole to a radical cation by RuIII with a rate constant k3 of 7 x 10(6) s-1. The cycle is completed by electron transfer from heme c FeII to the Trp-191 radical cation with a rate constant k4 of 6.1 x 10(4) s-1. The rate constant k4 decreases to 3.4 x 10(3) s-1 as the viscosity is increased to 84 cP, but the rate constants k1 and k3 remain the same. The results are consistent with a gating mechanism in which the Ru-27-Cc-CcP complex undergoes fluctuations between a major state A with the configuration of the hCc-CcP crystalline complex and a minor state B with the configuration of the yCc-CcP complex. The hCc-CcP complex, state A, has an inefficient pathway for electron transfer from heme c to the Trp-191 indolyl radical cation with a distance of 20.5 A and a predicted value of 5 x 10(2) s-1 for k4A. The observed rate constant k4 is thus gated by the rate constant ka for conversion of state A to state B, where the rate of electron transfer k4B is expected to be 2 x 10(6) s-1. The temperature dependence of k4 provides activation parameters that are consistent with the proposed gating mechanism. These studies provide evidence that configurational gating does not control electron transfer in the physiological yCc-CcP complex, but is required in the nonphysiological hCc-CcP complex.

2,2'-Dipyridyl↗

PubMed1999

NB1-C16-insulin: site-specific synthesis, purification, and biological activity.

PURPOSE: To develop a simple and efficient method for the synthesis and purification of NB1-lipid-modified-insulin without the use of protecting agents. METHODS: Bovine insulin was allowed to react with cis-9-hexadecenal in an aqueous-organic medium in the presence of NaBH3CN at room temperature overnight. HPLC and ESI LC/MS coupled with dithiothreitol and trypsin treatment were employed for product identification and optimization. The product was purified by a differential C18 solid-phase extraction. The biological effects of the modified insulin were evaluated by receptor binding assay and hypoglycemic effect measurement. RESULTS: NB1-cis-9-hexadecenyl insulin was synthesized by a one-step reductive alkylation in sodium salicylate and isopropanol solution in high yield (80%). The site selectivity and yield of the reaction were found to be affected by pH, medium, and insulin-to-aldehyde ratio. After solid phase extraction, the purity was found to be approximately 98%. This derivative showed a Kd to the insulin receptor of 5.72x10(-9) M and a significantly slower glucose lowering rate than insulin. CONCLUSIONS: NB1-hexadecenyl insulin was synthesized by reductive alkylation without the use of protective agents in high yield. NB1-hexadecenyl insulin retained significant binding affinity to insulin receptor and showed a pronounced hypoglycemic effect.

Alkylation↗

PubMed1999

[Identification of mutations of SRD5A2 gene and SRY gene in patients with hypospadias].

OBJECTIVE: To identify possible molecular mechanism underlined hypospadias and any relationship of the mutations of SRD5A2 gene and SRY gene to hypospadias. METHODS: Twenty-three blood samples from the patients with hypospadias were obtained from Aug.1996 to Jan. 1998. DNA was extracted from blood leukocytes. Exons 1 to 5 of the SRD5A2 gene and exon of the SRY gene were amplified by PCR. Mutation detection was performed using PCR-SSCP/silver staining and direct DNA sequencing. RESULTS: In 3 cases (named 5R2-China-1, 5R2-China-2, and 5R2-China-3), DNA sequencing revealed that a homozygous change from nucleotide G to A occurred in 5R2-China-1 and 2, leading to a substitution of glutamine to arginine in the codon 227(Arg 227 to Gln). In the third patient (5R2-China-3), DNA sequencing revealed two different heterozygous mutations(Arg 227 to Gln, Phe 186 to Leu) in exon 4 of the SRD5A2 gene. No mutation of SRY gene was found in all patients. CONCLUSION: The mutation of SRD5A2 gene affects the differentiation of the external genitalia and may play a role in the etiology of hypospadias. Since no mutation of SRY gene was found, this study might suggest that the mutation of SRY gene is not an important event in hypospadias. Codon 227 is a hotspot site of mutation within the gene. The mutation of codon 186(Phe 186Leu) represents a new form of SRD5A2 mutation.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗

PubMed1998

[Apoptosis and expression of PCNA in superficial transitional cell bladder cancer as related to recurrence].

OBJECTIVE: To determine the expression of apoptosis and PCNA in superficial transitional cell bladder cancer and to evaluate its significance as a recurrence marker. METHOD: 87 cases of superficial transitional cell bladder cancer were analysed for the expression of apoptosis and PCNA by using the 3-end labeling method of DNA and immunohistochemical staining in tissue sections. The apoptotic indexes and PCNA indexes were respectively the percentages of apoptotic cells or PCNA positive cells among tumor cells. RESULT: The end labeling method allowed a precise evaluation of the expression of apoptosis. Apoptosis occurred in 89.6% of superficial transitional cell bladder cancer. In 17 patients who suffered from tumor recurrence, the ratio of apoptosis indexes to PCNA indexes was significantly higher than that in those without recurrence (P < 0.01). CONCLUSION: The ratio of apoptosis indexes to PCNA indexes was believed to be related to tumor recurrence and might be of value in the assessment of recurrence of superficial transitional cell bladder cancer.

Adult↗

PubMed1998

Cloning and characterization of Schistosoma mansoni fructose-1,6-bisphosphate aldolase isoenzyme.

A Schistosoma mansoni cercarial cDNA expression library, constructed in lambda gt11, was screened using the IgG fraction of sera taken from rabbits vaccinated with irradiated cercariae. A positive cDNA clone (1,431 base pairs) was selected and characterized. The amino acid sequence predicted from the cDNA sequence identified a polypeptide of 363 amino acids that showed significant homology to different family members of the enzyme fructose-1,6-bisphosphate aldolase (EC 1.4.2.13). The identity was 66% and 65% with human C and A isoenzymes, respectively. Active sites and substrate-binding determinant analysis suggest that the isolated enzyme in terms of function resembles type A aldolase. The recombinant protein expressed in the vector pGEX-2T was found to be active enzymatically. Antibodies raised against the purified recombinant protein recognized a 40-kDa band in extracts from cercariae, schistosomula (5 and 25 days), adult worms, and eggs. Using immunocytochemistry, aldolase localized to the tegumental region of the adult worms.

Amino Acid Sequence↗

PubMed1997

Schistosoma mansoni: the developmental regulation and immunolocalization of antioxidant enzymes.

Antioxidant enzymes from S. mansoni, cytosolic Cu-Zn superoxide dismutase (CT-SOD), signal-peptide-containing SOD (SP-SOD), glutathione peroxidase (GPX), and glutathione transferase (GST) were compared for their relative levels of transcript expression throughout development in a semiquantitative reverse transcriptase-polymerase chain reaction assay. All of the antioxidant enzymes exhibited a similar pattern of developmental regulation. Adult worms have the highest level of specific mRNA compared with larval stages. GST shows the highest level of expression, being approximately 10-fold more abundant than CT-SOD and SP-SOD and 100-fold more abundant than GPX. This order of expression was nearly consistent for all the developmental stages studied. To localize the antioxidant enzymes, immunofluorescence staining was performed on 3-hr schistosomula and adult worms. GPX, SP-SOD, and CT-SOD were all found to be associated with the adult tegument and gut epithelium. SP-SOD was also associated with organelle and cell membranes of parenchymal cells and interestingly with the spines of adult worms. Schistosomula, on the other hand, showed little immunofluorescence. These studies further demonstrate the developmental regulation of antioxidant enzymes and localize them to the host-parasite interface, supporting the notion that they have a role in allowing adult worms to evade immune attack.

Animals↗

PubMed1996

Control of formation and dissociation of the high-affinity complex between cytochrome c and cytochrome c peroxidase by ionic strength and the low-affinity binding site.

A new ruthenium photoreduction technique was used to measure the formation and dissociation rate constants kf and kd of the high-affinity complex between yeast iso-1-cytochrome c (yCc) and cytochrome c peroxidase compound I (CMPI) over a wide range of ionic strength. These studies utilized Ru-39-Cc, which contains trisbipyridylruthenium attached to the cysteine residue in the H39C, C102T variant of yCc, and has the same reactivity with CMPI as native yCc. kd and kf were measured by photoreducing a small concentration of Ru-39-Cc in the presence of the oxidized yCcIII: CMPI complex, which must dissociate before Ru-39-CcII can bind to CMPI and reduce the radical action. The value of kd for the 1:1 high-affinity complex is very small at low ionic strength, < 5 s-1 but is increased significantly by binding yCc to a second low-affinity site. However, the low-affinity yCc binding site is not active in direct electron transfer to either the radical cation or the oxyferryl heme in CMPI, and is too weak to play a role in the kinetics at ionic strengths above 70 mM. The value of kd increases to 4000 s-1 at 150 mM ionic strength, while kf decreases from > 3 x 10(9) M-1 s-1 at low ionic strength to 1.3 x 10(9) M-1 s-1 at 150 mM ionic strength. These studies indicate that the rate-limiting step in enzyme turnover is product dissociation below 150 mM ionic strength and intracomplex electron transfer to the oxyferryl heme at higher ionic strength. The interaction between yCc and CcP is optimized at physiological ionic strength to provide the largest possible complex formation rate constant kf without allowing product dissociation to be rate-limiting. The effects of surface mutations on the kinetics provided evidence that the high-affinity binding site used for the reaction in solution is similar to the one identified in the yCc:CcP crystal structure.

Binding Sites↗

PubMed1996

Design of a ruthenium-cytochrome c derivative to measure electron transfer to the radical cation and oxyferryl heme in cytochrome c peroxidase.

A new ruthenium-labeled cytochrome c derivative was designed to measure the actual rate of electron transfer to the Trp-191 radical cation and the oxyferryl heme in cytochrome c peroxidase compound I {CMPI(FeIV = O,R.+)}. The H39C,C102T variant of yeast iso-1-cytochrome c was labeled at the single cysteine residue with a tris (bipyridyl)ruthenium(II) reagent to form Ru-39-Cc. This derivative has the same reactivity with CMPI as native yCc measured by stopped-flow spectroscopy, indicating that the ruthenium group does not interfere with the interaction between the two proteins. Laser excitation of the 1:1 Ru-39-Cc-CMPI complex in low ionic strength buffer (2 mM phosphate, pH 7) resulted in electron transfer from RuII* to heme c FeIII with a rate constant of 5 x 10(5) s-1, followed by electron transfer from heme c Fe II to the Trp-191 indolyl radical cation in CMPI(FeIV = O,R*+) with a rate constant of k(eta) = 2 x 10(6) s-1. A subsequent laser flash led to electron transfer from heme c to the oxyferryl heme in CMPII-(FeIV = O,R) with a rate constant of k(etb) = 5000 s-1. The location of the binding domain was determined using a series of surface charge mutants of CcP. The mutations D34N, E290N, and A193F each decreased the values of k(eta) and k(etb) by 2-4-fold, consistent with the use of the binding domain identified in the crystal structure of the yCc-CcP complex for reduction of both redox centers [Pelletier, H., & Kraut, J. (1992) Science 258, 1748-1755]. A mechanism is proposed for reduction of the oxyferryl heme in which internal electron transfer in CMPII(FeIV = O,R) leads to the regeneration of the radical cation in CMPII-(FeIII,R*+), which is then reduced by yCcII. Thus, both steps in the complete reduction of CMPI involve electron transfer from yCcII to the Trp-191 radical cation using the same binding site and pathway. Comparison of the rate constant k(eta) with theoretical predictions indicate that the electron transfer pathway identified in the crystalline yCc-CcP complex is very efficient. Stopped-flow studies indicate that native yCcII initially reduces the Trp-191 radical cation in CMPI with a second-order rate constant ka, which increases from 1.8 x 10(8) M-1 s-1 at 310 mM ionic strength to > 3 x 10(9) M-1 s-1 at ionic strengths below 100 mM. A second molecule of yCcII then reduces the oxyferryl heme in CMPII with a second-order rate constant kb which increases from 2.7 x 10(7) M-1 s-1 at 310 mM ionic strength to 2.5 x 10(8) M-1 s-1 at 160 mM ionic strength. As the ionic strength is decreased below 100 mM the rate constant for reduction of the oxyferryl heme becomes progressively slower as the reaction is limited by release of the product yCcIII from the yCcIII-CMPII complex. Both ruthenium photoreduction studies and stopped-flow studies demonstrate that the Trp-191 radical cation is the initial site of reduction in CMPI under all conditions of ionic strength.

2,2'-Dipyridyl↗