Review
The journey from NADPH-cytochrome P450 oxidoreductase to nitric oxide synthases☆

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Abstract

This mini-review will reflect the perspective of its author on two fields of research, which have merged as the result of the insights of investigators whose work has influenced both areas immeasurably. It cannot be overlooked, however, that the research activities of many during a period of over five decades have produced the chemical and biological bases for the exciting discoveries now encompassing the cytochromes P450 and their redox partners, and the three isoforms of nitric oxide synthase as they function in their respective biological milieux. Following the remarkable discovery that, indeed, molecular oxygen can be adducted to organic molecules by enzymatic systems and that such processes require a supply of reducing equivalents, it is the purpose of this review to provide a chart, with some of its detours, of the road that followed in the pursuit of interesting biological phenomena involving these two major oxygenation systems. It is not intended to be a balanced review and apologies must be offered in advance to those whose contributions may be overlooked or simply were not directly germane to the development of the author’s journey.

Section snippets

Discovery and early history of NADPH-cytochrome c (P450) reductase

The discovery of TPNH (NADPH)-cytochrome c reductase by Horecker in 1950 [1] set into motion over half a century of studies to determine the intracellular localization and biological function of this enzyme. These early studies by Horecker reported the isolation and purification of this enzyme from a whole liver acetone powder, which did not permit the localization of the enzyme to any subcellular compartment. Later, in 1956 and 1957, Strittmatter and Velick defined NADH-mediated activities in

Mechanism studies of NADPH-cytochrome c oxidoreductase as a flavoprotein

Much of the groundwork for mechanism studies of the NADPH-specific microsomal reductase was laid by the early experiments of Horecker [1], Williams and Kamin [5], and Phillips and Langdon [6]. The catalytically determined Km values for NADPH closely corresponded with those determined by Cohen and Estabrook [19] for monooxygenase reactions catalyzed by liver microsomes.

In the process of studying the steapsin-solubilized, purified reductase, lacking its N-terminal hydrophobic anchor required for

Discovery of the physiological role(s) of microsomal NADPH-cytochrome c oxidoreductase

As previously mentioned, Phillips and Langdon [6], La Du et al. [7], and Gillette et al. [8] had already speculated about the role of microsomal TPNH (NADPH)-cytochrome c reductase in other than the reduction of this mitochondrial cytochrome. The inhibition of microsomal monomethyl-4-aminoantipyrine demethylation by cytochrome c[8] predated the discovery of cytochromes P450, later shown to be involved in the oxidative demethylation (monooxygenation) of this substrate. In 1955, Klingenberg [33]

Reconstitution of the cytochrome P450-mediated monooxygenation systems in vitro

Although other studies had strongly implicated the interaction between CYPOR and microsomal cytochrome P450 in situ, very convincing evidence for this relationship was obtained with the reconstitution experiments of Lu and Coon [49] in which they were able to demonstrate the ω-hydroxylation of lauric acid with purified components. This report led the way to reconstitution of other systems when it became obvious that there were many cytochromes P450, even in liver microsomes, which needed to be

Evidence for convergent evolution of the diflavin enzymes from ferredoxin-NADP+ reductase and flavodoxins

It was first proposed by Porter and Kasper [51], [52] that NADPH-cytochrome P450 oxidoreductase (CYPOR) evolved from the ancestral genes coding for bacterial flavodoxins (FMN) and ferredoxin-NADP+ reductase. Although at the time of this work, no structure of CYPOR was available, the authors compared the sequences of ferredoxin-NADP+ reductase and NADH-cytochrome b5 reductase to glutathione reductase, of which the crystal structure was known, for FAD and cofactor binding similarities. The

The emergence of the nitric oxide synthases and their physiological significance

As early as 1916, Mitchell et al. [56] found that the diets of rats, pigs, and humans could not account for the amount of nitrogen oxides (NO3−) excreted in urine but there was no proof presented for metabolic production of such products. When Tannenbaum and colleagues [57], [58] showed that 15N-labeled precursors were converted to labeled nitrate metabolites, this proved that such metabolism occurred. These workers [58] showed that the source of these labeled metabolites was not bacterial by

Discovery of the sequence similarity of the C-terminal flavoprotein domain of neuronal nitric oxide synthase with NADPH-cytochrome P450 reductase

When Solomon Snyder’s laboratory reported the 58% sequence similarity between CYPOR and the C-terminal 641 amino acids of rat neuronal nitric oxide synthase [32], a new mammalian family of FAD- and FMN-containing enzymes was introduced. Other laboratories reported that nitric oxide synthases (NOSs) contained stoichiometric amounts of both FAD and FMN [66], [67], [68], [69] but no one had pursued the possibility of sequence similarity to known enzymes. In addition, because nitric oxide synthases

Discovery of heme in the oxygenase domains of nitric oxide synthases

Masters and her colleagues, having obtained the stably transfected clone of rat neuronal nitric oxide synthase (nNOS) in human kidney 293 cells from Solomon Snyder’s laboratory, invested the effort to isolate the enzyme from these cells in large quantities. In the meantime, other laboratories were conducting similar experiments on neuronal and/or inducible NOS enzymes. As a result, four different laboratories simultaneously demonstrated that these isoforms of NOS contained iron protoporphyrin

Cloning and expression of intact NOS isoforms in heterologous expression systems and subsequent purification and characterization

In order to pursue structure–function and biophysical experiments on the NOS isoforms, without the labor-intensive and expensive handling of mammalian cells in culture, several laboratories attempted to express the NOS isoforms in heterologous expression systems. Several publications appeared in the literature from the Moncada [88] and Marletta [79] laboratories describing the heterologous expression of nNOS cDNA in Spodoptera frugiperda (Sf-21) insect cells using a baculovirus vector but the

Exploration of the roles of the cofactor-binding domains of nitric oxide synthase isoforms in the regulation of activity: intrinsic regulation within the flavoprotein C-termini

Comparison of the sequences of the NO synthases by Salerno et al. [92] revealed very striking and important differences between the constitutive NOS isoforms and the inducible isoform (iNOS) and CYPOR. By sequence alignment, the insertion of approximately 45 residues, found to be located within the FMN-binding domain of both nNOS and eNOS, is notably absent in dozens of flavodoxins to which they were compared [92]. In order to demonstrate the ability of these additional residues to act as

Summary and conclusions

The ribbon structure of CYPOR from Wang et al. ([53]; Fig. 1A; blue) can be represented as a convergence of the structures of plant ferredoxin-NADP+ reductase (FNR) containing the FAD- and NADPH-binding sites (Fig. 1A; lower left, maroon) and bacterial flavodoxin (Fig. 1A; upper right, rose pink), as first proposed by Porter and Kasper [51], [52] based on sequence comparisons. In the development of mammalian enzymes, the utilization of the basic FAD- and NADPH-binding motifs of plant FNR and

Acknowledgments

This review would not have been possible without the dedication of a wonderful group of laboratory colleagues. These include: Drs. Linda Roman, Pavel Martásek, Satya Panda, Christopher Marohnic, Yingtong Gao, and Mark Panda, and the capable technical expertise of Senior Research Associates, Thomas Shea and Karen McKammon. I cannot forget to recognize the able oversight, daily support, and administrative help of Ms. Debbie Yrle. This review is dedicated to Dr. Osamu Hayaishi for his seminal

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    ☆

    Supported by National Institutes of Health Grant Nos. HL30050, GM52419, and GM31296 and Robert A. Welch Foundation Grant No. AQ-1192 to B.S.S.M.

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